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1.  YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer 
Stem cells (Dayton, Ohio)  2014;32(6):1437-1450.
There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated hTERT activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16INK4a-mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.
PMCID: PMC4321723  PMID: 24648416
Neoplastic cell transformation; Epigenetics; Breast cancer; Cancer stem cells; Human mammary epithelial cells (HMEC); Y-box binding protein-1 (YB-1)
2.  [No title available] 
PMCID: PMC3901373  PMID: 24420905
3.  [No title available] 
PMCID: PMC3947140  PMID: 24123810
4.  [No title available] 
PMCID: PMC3947148  PMID: 24123785
5.  [No title available] 
PMCID: PMC3961005  PMID: 24123709
6.  [No title available] 
PMCID: PMC4011928  PMID: 24420907
7.  SDF1 promotes CSPC quiescence via CK1α and GSK3β 
Stem cells (Dayton, Ohio)  2014;32(2):487-499.
A population of c-kit+ cardiac stem/progenitor cells (CSPC) has been identified in the heart and shown to contribute to myocardial regeneration after infarction. Previously, we have shown the chemokine, stromal cell derived factor 1α (SDF1) is necessary for the myocardial response to infarction where chronic infusion of the CXCR4 antagonist, AMD3100, exacerbated MI. Notably, AMD3100 increased CSPC proliferation. The effect of SDF1 on CSPC proliferation was further investigated in primary cultures of magnetically sorted c-kit+ CSPCs. SDF1 facilitated CSPC quiescence by blocking cell cycle progression at the G0 to G1 transition. SDF1 decreased casein kinase 1α (CK1α) consequently attenuating β-catenin phosphorylation, destabilization, and degradation. Increased levels of β-catenin with SDF1 were effective, increasing TCF/LEF reporter activity. SDF downregulation of CK1α was dependent on proteasomal degradation and decreased mRNA expression. CK1α siRNA knockdown verified SDF1-dependent CSPC quiescence requires CK1α downregulation and stablilization of β-catenin. Conversely, β-catenin knockdown increased CSPC proliferation. SDF1 also increased GSK3β Y216 phosphorylation responsible for increased activity. SDF1 mediated CK1α downregulation and increase in GSK3β activity affected cell cycle through Bmi-1 downregulation, increased cyclin D1 phosphorylation, and decreased cyclin D1 levels. In conclusion, SDF1 exerts a quiescent effect on resident c-kit+ CSPCs by decreasing CK1α levels, increasing GSK3β activity, stabilizing β-catenin, and affecting regulation of the cell cycle through Bmi-1 and cyclin D1. SDF1 dependent quiescence is an important factor in stem and progenitor cell preservation under basal conditions, however, with stress or injury in which SDF1 is elevated, quiescence may limit expansion and contribution to myocardial regeneration.
PMCID: PMC4161231  PMID: 24038789
Stromal cell derived factor 1α; CXCR4; Cell cycle; Casein kinase 1α; β-Catenin; Glycogen Synthase Kinase3β
8.  Dynamic migration and cell-cell interactions of early reprogramming revealed by high resolution time-lapse imaging 
Stem cells (Dayton, Ohio)  2013;31(5):895-905.
Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multi-color time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as five minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct 2-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate the requirement of E-cadherin for proper cellular interactions from an early stage of reprogramming, including the 2-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.
PMCID: PMC4309553  PMID: 23335078
9.  Expression of Podocalyxin separates the hematopoietic and vascular potentials of mouse ES cell-derived mesoderm 
Stem cells (Dayton, Ohio)  2014;32(1):191-203.
In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5 and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.
PMCID: PMC3908821  PMID: 24022884
mouse embryonic stem cells; pluripotent cells; mesoderm induction; gastrulation; hematopoiesis; endothelial cell; vascular development; Flk1; Podocalyxin
10.  Oct4 maintains the pluripotency of human embryonic stem cells by inactivating p53 through Sirt1-mediated deacetylation 
Stem cells (Dayton, Ohio)  2014;32(1):157-165.
Oct4 is critical to maintain the pluripotency of human embryonic stem cells (hESCs), however, the underlying mechanism remains to be fully understood. Here we report that silencing of Oct4 in hESCs leads to the activation of tumor suppressor p53, inducing the differentiation of hESCs since acute disruption of p53 in p53 conditional knockout (p53CKO) hESCs prevents the differentiation of hESCs after Oct4 depletion. We further discovered that the silencing of Oct4 significantly reduces the expression of Sirt1, a deacetylase known to inhibit p53 activity and the differentiation of ESCs, leading to increased acetylation of p53 at lysine 120 and 164. The importance of Sirt1 in mediating Oct4-dependent pluripotency is revealed by the finding that the ectopic expression of Sirt1 in Oct4-silenced hESCs prevents p53 activation and hESC differentiation. In addition, employing knock-in approach, we revealed that the acetylation of p53 at lysine 120 and 164 is required for both stabilization and activity of p53 in hESCs. In summary, our findings reveal a novel role of Oct4 in maintaining the pluripotency of hESCs by suppressing pathways that induce differentiation. Considering that p53 suppresses pluripotency after DNA damage response in ESCs, our findings further underscore the stringent mechanism to coordinate DNA damage response pathways and pluripotency pathways in order to maintain the pluripotency and genomic stability of hESCs.
PMCID: PMC3947311  PMID: 24038750
pluripotency; differentiation; human ES cells; DNA damage response; p53; acetylation
11.  Human Mesenchymal Stem Cell Microvesicles for Treatment of E.coli Endotoxin-Induced Acute Lung Injury in Mice 
Stem cells (Dayton, Ohio)  2014;32(1):116-125.
We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following E.coli endotoxin-induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble factors such as keratinocyte growth factor (KGF). Recently, MSC were found to release microvesicles (MV) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. The current studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E.coli endotoxin-induced ALI in C57BL/6 mice. The intra-tracheal instillation of MVs improved several indices of ALI at 48 h. Compared to endotoxin-injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein-2 levels in the BAL fluid by 73% and 49% respectively, demonstrating a reduction in inflammation. KGF siRNA-pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC derived microvesicles were therapeutically effective following E.coli endotoxin-induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus.
PMCID: PMC3947321  PMID: 23939814
Acute Lung injury; Keratinocyte growth factor; Lipopolysaccharide; Mesenchymal stem cell; Microvesicles
12.  A high Notch pathway activation predicts response to γ secretase inhibitors in proneural subtype of glioma tumor initiating cells 
Stem cells (Dayton, Ohio)  2014;32(1):301-312.
Genomic, transcriptional, and proteomic analyses of brain tumors reveal subtypes that differ in pathway activity, progression, and response to therapy. However, a number of small molecule inhibitors under development vary in strength of subset and pathway-specificity, with molecularly targeted experimental agents tending toward stronger specificity. The Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes. We investigated the effects of Notch pathway inhibition in glioma tumor initiating cell (GIC, hereafter GIC) populations using γ secretase inhibitors. Drug cytotoxicity testing of 16 GICs showed differential growth responses to the inhibitors, stratifying GICs into responders and non-responders. Responder GICs had an enriched proneural gene signature in comparison to non-responders. Also gene set enrichment analysis revealed 17 genes set representing active Notch signaling components NOTCH1, NOTCH3, HES1, MAML1, DLL-3, JAG2 etc., enriched in responder group. Analysis of TCGA expression data set identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation suggesting γ-secretase inhibitors might be of potential value to treat that particular group of proneural GBM. Inhibition of Notch pathway by γ-secretase inhibitor treatment attenuated proliferation and self-renewal of responder GICs and induces both neuronal and astrocytic differentiation. In vivo evaluation demonstrated prolongation of median survival in an intracranial mouse model. Our results suggest that proneural GBM characterized by high Notch pathway activation may exhibit greater sensitivity to γ-secretase inhibitor treatment, holding a promise to improve the efficiency of current glioma therapy.
PMCID: PMC3947402  PMID: 24038660
Notch activation; proneural genes; γ secretase inhibitors; Glioma
13.  Pax6 Mediates β-Catenin Signaling for Self-Renewal and Neurogenesis by Neocortical Radial Glial Stem Cells 
Stem cells (Dayton, Ohio)  2014;32(1):45-58.
The Wnt/β-catenin pathway is a critical stem cell regulator and plays important roles in neuroepithelial cells during early gestation. However, the role of Wnt/β-catenin signaling in radial glia, a major neural stem cell population expanded by midgestation, remains poorly understood. This study shows that genetic ablation of β-catenin with hGFAP-Cre mice inhibits neocortical formation by disrupting radial glial development. Reduced radial glia and intermediate progenitors are found in the β-catenin-deficient neocortex during late gestation. Increased apoptosis and divergent localization of radial glia in the subventricular zone are also observed in the mutant neocortex. In vivo and in vitro proliferation and neurogenesis as well as oligodendrogenesis by cortical radial glia or by dissociated neural stem cells are significantly defective in the mutants. Neocortical layer patterning is not apparently altered, while astrogliogenesis is ectopically increased in the mutants. At the molecular level, the expression of the transcription factor Pax6 is dramatically diminished in the cortical radial glia and the sphere-forming neural stem cells of β-catenin-deficient mutants. Chromatin immunoprecipitation and luciferase assays demonstrate that β-catenin/Tcf complex binds to Pax6 promoter and induces its transcriptional activities. The forced expression of Pax6 through lentiviral transduction partially rescues the defective proliferation and neurogenesis by β-catenin-deficient neural stem cells. Thus, Pax6 is a novel downstream target of the Wnt/β-catenin pathway, and β-catenin/Pax6 signaling plays critical roles in self-renewal and neurogenesis of radial glia/neural stem cells during neocortical development.
PMCID: PMC3963407  PMID: 24115331
Neural stem cells; Radial glia; Wnt/β-catenin; Pax6; Proliferation; Neurogenesis
14.  The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells 
Stem cells (Dayton, Ohio)  2014;32(1):135-144.
Cancer stem cells (CSCs) or tumor-initiating cells, similar to normal tissue stem cells, rely on developmental pathways, such as the Notch pathway, to maintain their stem cell state. One of the regulators of the Notch pathway is Musashi-1, a mRNA-binding protein. Musashi-1 promotes Notch signaling by binding to the mRNA of Numb, the negative regulator of Notch signaling, thus preventing its translation. Cancer stem cells have also been shown to down-regulate their 26S proteasome activity in several types of solid tumors, thus making them resistant to proteasome-inhibitors used as anti-cancer agents in the clinic. Interestingly, the Notch pathway can be inhibited by proteasomal degradation of the Notch intracellular domain (Notch-ICD), therefore down-regulation of the 26S proteasome activity can lead to stabilization of Notch-ICD. Here we present evidence that the down-regulation of the 26S proteasome in CSCs constitutes another level of control by which Musashi-1 promotes signaling through the Notch pathway and maintenance of the stem cell phenotype of this subpopulation of cancer cells. We demonstrate that Musashi-1 mediates the down-regulation of the 26S proteasome by binding to the mRNA of NF-YA, the transcriptional factor regulating 26S proteasome subunit expression, thus providing an additional route by which the degradation of Notch-ICD is prevented, and Notch signaling is sustained.
PMCID: PMC3968686  PMID: 24022895
Breast cancer stem cells; glioma stem cells; radiation; Notch; proteasome; Musashi; NF-Y
15.  Pten Loss in Olig2 Expressing Neural Progenitor Cells and Oligodendrocytes Leads to Interneuron Dysplasia and Leukodystrophy 
Stem cells (Dayton, Ohio)  2014;32(1):313-326.
Therapeutic modulation of PI3K/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this we created Olig2-cre:Ptenfl/fl mice which showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Ptenfl/fl animals progressively developed CNS white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration and death by nine months. In contrast, during immediate post-natal development oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Ptenfl/fl mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor, NVP-BKM120, blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary, our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However, such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development.
PMCID: PMC4096461  PMID: 24395742
hypermyelination; Olig2; Pten; interneurons; leukodystrophy
16.  Generation of Functional, Antigen-Specific CD8+ Human T Cells from Cord-Blood Stem Cells using Exogenous Notch and Tetramer-TcR Signaling 
Stem cells (Dayton, Ohio)  2014;32(1):93-104.
In vitro differentiation of mouse and human stem cells into early T cells has been successfully demonstrated using artificial Notch signaling systems. However, generation of mature, antigen-specific, functional T cells, directly from human stem cells has remained elusive, except when using stromal co-culture of stem cells retrovirally transfected with antigen-specific T cell receptors (TCRs). Here we show that human umbilical cord blood (UCB)-derived CD34+CD38−/low hematopoietic stem cells (HSCs) can be successfully differentiated into functional, antigen-specific cytotoxic CD8+ T cells without direct stromal co-culture or retroviral TCR transfection. Surface-immobilized Notch ligands (DLL1) and stromal cell conditioned medium successfully induced the development of CD1a+CD7+ and CD4+CD8+ early T cells. These cells, upon continued culture with cytomegalovirus (CMV) or Influenza-A virus epitope-loaded HLA-A*0201 tetramers, resulted in the generation of a polyclonal population of CMV-specific or Influenza-specific CD8+ T cells respectively. Upon further activation with antigen-loaded target cells, these antigen-specific, stem cell-derived T cells exhibited cytolytic functionality, specifically CD107a surface mobilization, IFNγ production, and Granzyme B secretion. Such scalable, in vitro generation of functional, antigen-specific human T cells from human stem cells could eventually provide a readily available cell source for adoptive transfer immunotherapies and also allow better understanding of human T cell development.
PMCID: PMC4193361  PMID: 23939944
CD34+ cells; adult hematopoietic stem cells; human cord blood; Cytotoxic T cells
17.  Current Status of Stem Cells and Regenerative Medicine in Lung Biology and Diseases 
Stem cells (Dayton, Ohio)  2014;32(1):16-25.
Lung diseases remain a significant and devastating cause of morbidity and mortality worldwide. In contrast to many other major diseases, lung diseases notably chronic obstructive pulmonary diseases (COPD), including both asthma and emphysema, are increasing in prevalence and COPD is expected to become the 3rd leading cause of disease mortality worldwide by 2020. New therapeutic options are desperately needed. A rapidly growing number of investigations of stem cells and cell therapies in lung biology and diseases as well as in ex vivo lung bioengineering have offered exciting new avenues for advancing knowledge of lung biology as well as providing novel potential therapeutic approaches for lung diseases. These initial observations have led to a growing exploration of endothelial progenitor cells and mesenchymal stem (stromal) cells in clinical trials of pulmonary hypertension and chronic obstructive pulmonary disease (COPD) with other clinical investigations planned. Ex vivo bioengineering of the trachea, larynx, diaphragm, and the lung itself with both biosynthetic constructs as well as decellularized tissues have been utilized to explore engineering both airway and vascular systems of the lung. Lung is thus a ripe organ for a variety of cell therapy and regenerative medicine approaches. Current state-of-the-art progress for each of the above areas will be presented as will discussion of current considerations for cell therapy based clinical trials in lung diseases.
PMCID: PMC4208500  PMID: 23959715
Lung; lung regeneration; lung diseases; endogenous progenitor cell; mesenchymal stem cell; endothelial progenitor cell; embryonic stem cell; induced pluripotent stem cell; cell therapy; bioengineering
18.  Repression of Zeb1 and Hypoxia Cause Sequential MET and Induction of Aid, Oct4, and Dnmt1, Leading to Immortalization and Multipotential Reprogramming of Fibroblasts in Spheres 
Stem cells (Dayton, Ohio)  2013;31(7):1350-1362.
Here, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the EMT transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. And, hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor.
PMCID: PMC4265806  PMID: 23554223
19.  Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina 
Stem cells (Dayton, Ohio)  2011;29(6):972-980.
Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments utilizing stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with swine induced pluripotent stem cells (iPSC). Here, we subjected swine iPSC to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of RHO and ROM1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that swine iPSC can differentiate into photoreceptors in culture and these cells can integrate into the damaged swine neural retina thus laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.
PMCID: PMC4263955  PMID: 21491544
photoreceptor differentiation; swine retina; induced pluripotent stem cells; retinal transplantation
20.  Molecular Diversity Subdivides the Adult Forebrain Neural Stem Cell Population 
Stem cells (Dayton, Ohio)  2014;32(1):70-84.
Neural stem cells (NSCs) in the ventricular domain of the subventricular zone (V-SVZ) of rodents produce neurons throughout life while those in humans become largely inactive or may be lost during infancy. Most adult NSCs are quiescent, express glial markers, and depend on Notch signaling for their self-renewal and the generation of neurons. Using genetic markers and lineage tracing, we identified subpopulations of adult V-SVZ NSCs (type 1, 2, and 3) indicating a striking heterogeneity including activated, brain lipid binding protein (BLBP, FABP7) expressing stem cells. BLBP+ NSCs are mitotically active components of pinwheel structures in the lateral ventricle walls and persistently generate neurons in adulthood. BLBP+ NSCs express epidermal growth factor (EGF) receptor, proliferate in response to EGF, and are a major clonogenic population in the SVZ. We also find BLBP expressed by proliferative ventricular and sub-ventricular progenitors in the fetal and postnatal human brain. Loss of BLBP+ stem/progenitor cells correlates with reduced neurogenesis in aging rodents and postnatal humans. These findings of molecular heterogeneity and proliferative differences subdivide the NSC population and have implications for neurogenesis in the forebrain of mammals during aging.
PMCID: PMC4259462  PMID: 23964022
Neurogenesis; Subventricular zone; Neural stem cells; Aging
21.  Epigenetic re-programming of the Germ Cell Nuclear Factor gene is required for proper differentiation of induced pluripotent cells 
Stem cells (Dayton, Ohio)  2013;31(12):2659-2666.
Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that Germ Cell Nuclear Factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly re-programmed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not re-programmed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells re-capitulated the phenotype of Gcnf knock out (Gcnf−/−) ES cells. Re-introduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf−/− ES cells, rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely re-programmed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells.
PMCID: PMC3731425  PMID: 23495137
Gcnf; iPS cells; somatic cell reprogramming; epigenetics; stem cells
22.  MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles 
Stem cells (Dayton, Ohio)  2013;31(12):2737-2746.
To test, in vivo, the hypothesis that exosomes from multipotent mesenchymal stromal cells (MSCs) mediate microRNA 133b (miR-133b) transfer which promotes neurological recovery from stroke, we employed knock-in and knock-down technologies to up-regulate or down-regulate the miR-133b level in MSCs (miR-133b+MSCs or miR-133b−MSCs) and their corresponding exosomes, respectively. Rats were subjected to middle cerebral artery occlusion (MCAo) and were treated with naïve MSCs, miR-133b+MSCs, or miR-133b−MSC at one day after MCAo. Compared with controls, rats receiving naïve MSC treatment significantly improved functional recovery, and exhibited increased axonal plasticity and neurite remodeling in the ischemic boundary zone (IBZ) at day 14 after MCAo. The outcomes were significantly enhanced with miR-133b+MSC treatment, and were significantly decreased with miR-133b−MSC treatment, compared to naïve MSC treatment. The miR-133b level in exosomes collected from the cerebral spinal fluid was significantly increased after miR-133b+MSC treatment, and was significantly decreased after miR-133b−MSC treatment at day 14 after MCAo, compared to naïve MSC treatment. Tagging exosomes with green fluorescent protein demonstrated that exosomes-enriched extracellular particles were released from MSCs and transferred to adjacent astrocytes and neurons. The expression of selective targets for miR-133b, connective tissue growth factor and ras homolog gene family member A, were significantly decreased in the IBZ after miR-133b+MSC treatment, while their expression remained at similar elevated levels after miR-133b−MSC treatment, compared to naïve MSC treatment. Collectively, our data suggest that exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke.
PMCID: PMC3788061  PMID: 23630198
microRNA 133b; exosomes; multipotent mesenchymal stromal cells; neurite remodeling; stroke
23.  Gene regulatory networks mediating canonical Wnt signal directed control of pluripotency and differentiation in embryo stem cells 
Stem cells (Dayton, Ohio)  2013;31(12):2667-2679.
Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Further, canonical Wnt signaling up-regulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a MEK/ERK inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation.
PMCID: PMC3830733  PMID: 23505158
Mouse embryonic stem cells; Wnt; β-catenin; 2i; pluripotency; differentiation
Stem cells (Dayton, Ohio)  2013;31(12):2767-2778.
Tissue specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA)-injury and then returned to normal. The present study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:GFP model to purify basal cells that proliferated infrequently (GFPbright) or frequently (GFPdim) after NA-injury. Both populations contained TSC but TSC were 8.5-fold more abundant in the GFPbright population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally-differentiated basal cells. The ratio of TSC to UBP was 5:1 in the GFPbright population and 1:5 in the GFPdim population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFPbright and GFPdim populations and passaged the clones 7 times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFPdim lineage. Gene expression analysis showed that β-catenin and Notch pathway genes were differentially expressed in freshly-isolated TSC derived from GFPbright and GFPdim populations. We conclude that: 1) TSC and UPB are members of a single lineage; 2) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and 3) an interaction between the β-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.
PMCID: PMC3844014  PMID: 23712882
multipotential stem cells; unipotential basal progenitor; terminal differentiation; Wnt/β-catenin; Notch
25.  Engineering Myocardial Tissue: The Convergence of Stem Cells Biology and Tissue Engineering Technology 
Stem cells (Dayton, Ohio)  2013;31(12):2587-2598.
Advanced heart failure represents a leading public health problem in the developed world. The clinical syndrome results from the loss of viable and/or fully functional myocardial tissue. Designing new approaches to augment the number of functioning human cardiac muscle cells in the failing heart serve as the foundation of modern regenerative cardiovascular medicine. A number of clinical trials have been performed in an attempt to increase the number of functional myocardial cells by the transplantation of a diverse group of stem or progenitor cells. Although there are some encouraging suggestions of a small early therapeutic benefit, to date, no evidence for robust cell or tissue engraftment has been shown, emphasizing the need for new approaches. Clinically meaningful cardiac regeneration requires the identification of the optimum cardiogenic cell types and their assembly into mature myocardial tissue that is functionally and electrically coupled to the native myocardium. We here review recent advances in stem cell biology and tissue engineering and describe how the convergence of these two fields may yield novel approaches for cardiac regeneration.
PMCID: PMC3912205  PMID: 23843322

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