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1.  Locally-delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs 
Stem cells (Dayton, Ohio)  2009;27(8):1976-1986.
Ischemia is a potentially fatal medical event that is associated with as many as 30 percent of all deaths. Stem cell therapy offers significant therapeutic promise, but poor survival following transplantation to ischemic tissue limits its efficacy. Here we demonstrate that nanosphere-mediated growth factor delivery can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors per cell, and substantially improve therapeutic efficacy of hADSCs. In vitro, in hypoxic (1% oxygen) and serum-deprived conditions which simulate in vivo ischemia, fibroblast growth factor-2 (FGF2) significantly reduced hADSC apoptosis and enhanced angiogenic growth factor secretion. In vivo, hADSCs delivered intramuscularly into ischemic hindlimbs in combination with FGF2 resulted in significant improvements in limb survival and blood perfusion, as well as survival of the transplanted hADSCs and secretion of human angiogenic growth factors (i.e., vascular endothelial growth factor, hepatocyte growth factor, and FGF2). Interestingly, the majority of transplanted hADSCs were localized adjacent to the microvessels rather than being incorporated into them, suggesting that their major contribution to angiogenesis might be to increase paracrine secretion of angiogenic growth factors. This study demonstrates the potential of hADSCs in combination with growth factors for use in the treatment of ischemia.
doi:10.1002/stem.115
PMCID: PMC5215850  PMID: 19544425
Human adipose-derived stromal cell; Fibroblast growth factor-2; Local delivery; Paracrine effect; Hindlimb ischemia; Therapeutic angiogenesis
2.  Induced pluripotent stem cell-derived natural killer cells for treatment of ovarian cancer 
Stem cells (Dayton, Ohio)  2015;34(1):93-101.
Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB-) and NK cells derived from induced pluripotent stem cell (iPSC-) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of peripheral blood B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given 3 doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate anti-ovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.
doi:10.1002/stem.2230
PMCID: PMC4713309  PMID: 26503833
induced pluripotent stem cells; natural killer cells; ovarian cancer; immunotherapy
3.  Abrogation of age-induced microRNA-195 rejuvenates the senescent mesenchymal stem cells by reactivating telomerase 
Stem cells (Dayton, Ohio)  2015;34(1):148-159.
Previously, we reported that a novel sub-population of young mesenchymal stem cells (YMSCs) existed in old bone marrow, which possessed high anti-aging properties as well as excellent efficacy for cardiac repair. MicroRNAs (miRNAs) have emerged as key regulators in post-transcriptional gene expression programs, and however, it is unknown whether miRNAs directly control stem cell senescence. Here we present the first evidence that miR-195 overexpressed in old MSCs induces stem cell senescence deteriorating their regenerative ability by directly deactivating telomerase reverse transcriptase (Tert), and abrogation of miR-195 can reverse stem cell aging. MiRNAs profiling analysis in YMSCs and OMSCs by microarray showed that miR-140, miR-146a/b and miR-195 were significantly upregulated in OMSCs, which led us to hypothesize that these are age-induced miRNAs involved in stem cell senescence. Of these miRNAs, we found miR-195 directly targeted 3′-untranslated region of Tert gene by computational target prediction analysis and luciferase assay, and knockdown of miR-195 significantly increased Tert expression in OMSCs. Strikingly, miR-195 inhibition significantly induced telomere re-lengthening in OMSCs along with reduced expression of senescence-associated β-galactosidase. Moreover, silencing miR-195 in OMSCs by transfection of miR-195 inhibitor significantly restored anti-aging factors expression including Tert and Sirt1 as well as phosphorylation of Akt and FOXO1. Notably, abrogation of miR-195 markedly restored proliferative abilities in OMSCs. Transplantation of OMSCs with knocked out miR-195 reduced infarction size and improved LV function. In conclusion, rejuvenation of aged stem cells by miR-195 inhibition would be a promising autologous therapeutic strategy for cardiac repair in the elderly patients.
doi:10.1002/stem.2211
PMCID: PMC4797648  PMID: 26390028
Aging; stem cells; miR-195; telomerase; heart
4.  Functional Niche Competition Between Normal Hematopoietic Stem and Progenitor Cells and Myeloid Leukemia Cells 
Stem cells (Dayton, Ohio)  2015;33(12):3635-3642.
Hematopoietic stem and progenitor cells (HSPC) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells, however whether normal HSC and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the MLL-AF9 murine acute myeloid leukemia in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to anti-leukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation.
doi:10.1002/stem.2208
PMCID: PMC4713243  PMID: 26388434
bone marrow; niche; hematopoietic stem cells; acute myeloid leukemia; murine; competition
5.  Epigenetic control of Apolipoprotein E expression mediates gender-specific hematopoietic regulation 
Stem cells (Dayton, Ohio)  2015;33(12):3643-3654.
Epigenetic alterations play a central role in the control of normal and malignant blood cell development. We demonstrate here that expression of a truncated DNA methyltransferase 3B isoform DNMT3B7, which has been shown to alter cellular epigenetic patterns, decreases the overall number of hematopoietic stem and progenitor cells (HSPCs), and markedly diminishes blood cell reconstitution within the female hormonal microenvironment. Gene expression profiling of HSPCs isolated from DNMT3B7 transgenic embryos identified Apolipoprotein E (Apoe) as overexpressed. The CpG island controlling Apoe expression had lower levels of modified cytosines in DNMT3B7 transgenic HSPCs, corresponding with the observed increase in gene expression. Furthermore, we observed that spleens and bone marrows of female mice transplanted with DNMT3B7 transgenic HSPCs express very high levels of Apoe. Finally, the introduction of Apoe-overexpressing HSPCs into male recipients decreased bone marrow engraftment, recapitulating our original observations in female recipients. Our work reveals a dynamic interplay between the intrinsic epigenetic changes in HSPCs and extrinsic endocrine factors acting on these cells to regulate the efficiency of hematopoietic stem and progenitor cell engraftment and reconstitution. We have identified a novel mechanism by which gender-specific hormones modulate HSPC function, which could serve as a target for augmenting hematopoiesis in cases with limited HSC functionality.
doi:10.1002/stem.2214
PMCID: PMC4713251  PMID: 26417967
Hematopoiesis; Epigenetics; DNA methyltransferase 3B; Gender-specific hormones; Apolipoprotein E
6.  CHROMATIN REMODELING FACTOR BRG1 SUPPORTS THE EARLY MAINTENANCE AND LATE RESPONSIVENESS OF NESTIN-LINEAGE ADULT NEURAL STEM AND PROGENITOR CELLS 
Stem cells (Dayton, Ohio)  2015;33(12):3655-3665.
Insights from embryonic development suggest chromatin remodeling is important in adult neural stem cells (aNSCs) maintenance and self-renewal, but this concept has not been fully explored in the adult brain. To assess the role of chromatin remodeling in adult neurogenesis, we inducibly deleted Brg1 – the core subunit of SWI/SNF-like BAF chromatin remodeling complexes – in nestin-expressing aNSCs and their progeny in vivo and in culture. This resulted in abnormal adult neurogenesis in the hippocampus, which initially reduced hippocampal aNSCs and progenitor maintenance, and later reduced its responsiveness to physiological stimulation. Mechanistically, deletion of Brg1 appeared to impair cell cycle progression, which is partially due to elevated p53 pathway and p21 expression. Knockdown of p53 rescued the neurosphere growth defects caused by Brg1 deletion. Our results show that epigenetic chromatin remodeling (via a Brg1 and p53/p21-dependent process) determines the aNSCs and progenitor maintenance and responsiveness of neurogenesis.
doi:10.1002/stem.2215
PMCID: PMC4713255  PMID: 26418130
Neural stem cell; Adult stem cell; Cellular proliferation; Cre-loxP system; Flow cytometry; Epigenetics; Nervous System; Transgenic mouse
7.  CD150− side population defines leukemia stem cells in a BALB/C mouse model of CML and is depleted by genetic loss of SIRT1 
Stem cells (Dayton, Ohio)  2015;33(12):3437-3451.
Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here we show that lineage negative CD150− side population (CD150− SP), but not CD150+ SP, are CML LSCs in this model, although both CD150− SP and CD150+ SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study we demonstrate that SIRT1 homozygous knockout substantially reduces CD150− SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150−SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through acetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs.
doi:10.1002/stem.2218
PMCID: PMC4713280  PMID: 26466808
leukemia stem cells (LSCs); hematopoietic stem cells (HSCs); side population; SIRT1; Chronic myeloid leukemia (CML)
8.  Negative Regulation of miR-375 by Interleukin-10 Enhances Bone Marrow derived Progenitor Cell-mediated Myocardial Repair and Function after Myocardial Infarction 
Stem cells (Dayton, Ohio)  2015;33(12):3519-3529.
Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell based therapies. We have earlier reported that co-administration of IL-10 and BMPAC enhances cell survival and improves LV (LV) functions after AMI in mice. We hypothesized that IL-10 regulates miR-375 signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. MicroRNA-375 expression is significantly up regulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 KO mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knock down in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization and LV functions. Our in vitro studies revealed that knockdown of miR-375 enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase 1 (PDK-1) expression and PDK-1-mediated activation of PI3Kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.
doi:10.1002/stem.2121
PMCID: PMC4713300  PMID: 26235810
Bone marrow progenitor angiogenic cells; miRNA; inflammation; angiogenesis; left ventricular remodeling
9.  Transcriptome dynamics of developing photoreceptors in 3-D retina cultures recapitulates temporal sequence of human cone and rod differentiation revealing cell surface markers and gene networks 
Stem cells (Dayton, Ohio)  2015;33(12):3504-3518.
The derivation of three-dimensional (3-D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a GFP reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of post-mitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3-D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor NRL is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating co-expression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.
doi:10.1002/stem.2122
PMCID: PMC4713319  PMID: 26235913
Human Rod and Cone Photoreceptors; Stem Cells; Global Gene Profiling; Next Generation Sequencing; Three Dimensional Organoid Culture; Retina
10.  A mouse model of targeted Musashi1 expression in whole intestinal epithelium suggests regulatory roles in cell cycle and stemness 
Stem cells (Dayton, Ohio)  2015;33(12):3621-3634.
The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. In order to undertake a detailed study of Musashi1’s function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium.
Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1’s association with gut stem cell signature, cell cycle, DNA replication and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6 and Sox4.
In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle and DNA synthesis and repair.
doi:10.1002/stem.2202
PMCID: PMC4713339  PMID: 26303183
Intestinal epithelium; Musashi1; RNA binding protein; stem cells
11.  Pharyngeal satellite cells undergo myogenesis under basal conditions and are required for pharyngeal muscle maintenance 
Stem cells (Dayton, Ohio)  2015;33(12):3581-3595.
The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.
doi:10.1002/stem.2098
PMCID: PMC4713349  PMID: 26178867
pharynx; satellite cells; myofiber; myonuclear turnover; muscle maintenance
12.  Ataxia Telangiectasia Mutated Dysregulation Results in Diabetic Retinopathy 
Stem cells (Dayton, Ohio)  2015;34(2):405-417.
Ataxia telangiectasia mutated (ATM) acts as a defense against a variety of bone marrow (BM) stressors. We hypothesized that ATM loss in BM-hematopoietic stem cells (HSCs) would be detrimental to both HSC function and microvascular repair while sustained ATM would be beneficial in disease models of diabetes. Chronic diabetes represents a condition associated with HSC depletion and inadequate vascular repair. Gender mismatched chimeras of ATM−/− on wild type background were generated and a cohort were made diabetic using streptozotocin (STZ). HSCs from the STZ-ATM−/−chimeras showed (a) reduced self-renewal; (b) decreased long-term repopulation; (c) depletion from the primitive endosteal niche; (d) myeloid bias; and (e) accelerated diabetic retinopathy (DR). To further test the significance of ATM in hematopoiesis and diabetes, we performed microarrays on circulating angiogenic cells, CD34+ cells, obtained from a unique cohort of human subjects with long-standing (>40 years duration) poorly controlled diabetes that were free of DR. Pathway analysis of microarrays in these individuals revealed DNA repair and cell-cycle regulation as the top networks with marked upregulation of ATM mRNA compared with CD34+ cells from diabetics with DR. In conclusion, our study highlights using rodent models and human subjects, the critical role of ATM in microvascular repair in DR.
doi:10.1002/stem.2235
PMCID: PMC5125377  PMID: 26502796
Diabetic retinopathy; Ataxia telangiectasia mutated; Hematopoietic stem cells
13.  Concise Review: Measuring Physiological Responses of Human Pluripotent Stem Cell Derived Cardiomyocytes to Drugs and Disease 
Stem Cells (Dayton, Ohio)  2016;34(8):2008-2015.
Abstract
Cardiomyocytes from human pluripotent stem cells (hPSC) are of growing interest as models to understand mechanisms underlying genetic disease, identify potential drug targets and for safety pharmacology as they may predict human relevant effects more accurately and inexpensively than animals or other cell models. Crucial to their optimal use are accurate methods to quantify cardiomyocyte phenotypes accurately and reproducibly. Here, we review current methods for determining biophysical parameters of hPSC‐derived cardiomyocytes (hPSC‐CMs) that recapitulate disease and drug responses. Even though hPSC‐CMs as currently available are immature, various biophysical methods are nevertheless already providing useful insights into the biology of the human heart and its maladies. Advantages and limitations of assays currently available looking toward applications of hPSC‐CMs are described with examples of how they have been used to date. This will help guide the choice of biophysical method to characterize healthy cardiomyocytes and their pathologies in vitro. Stem Cells 2016;34:2008–2015
doi:10.1002/stem.2403
PMCID: PMC5113667  PMID: 27250776
Human; Pluripotent stem cells; Heart; Drug testing; Cardiac disease modelling; Cardiomyocyte maturation; Cardiomyocyte physiology
14.  Local CXCR4 Upregulation in the Injured Arterial Wall Contributes to Intimal Hyperplasia 
Stem Cells (Dayton, Ohio)  2016;34(11):2744-2757.
Abstract
CXCR4 is a stem/progenitor cell surface receptor specific for the cytokine stromal cell‐derived factor‐1 (SDF‐1α). There is evidence that bone marrow‐derived CXCR4‐expressing cells contribute to intimal hyperplasia (IH) by homing to the arterial subintima which is enriched with SDF‐1α. We have previously found that transforming growth factor‐β (TGFβ) and its signaling protein Smad3 are both upregulated following arterial injury and that TGFβ/Smad3 enhances the expression of CXCR4 in vascular smooth muscle cells (SMCs). It remains unknown, however, whether locally induced CXCR4 expression in SM22 expressing vascular SMCs plays a role in neointima formation. Here, we investigated whether elevated TGFβ/Smad3 signaling leads to the induction of CXCR4 expression locally in the injured arterial wall, thereby contributing to IH. We found prominent CXCR4 upregulation (mRNA, 60‐fold; protein, 4‐fold) in TGFβ‐treated, Smad3‐expressing SMCs. Chromatin immunoprecipitation assays revealed a specific association of the transcription factor Smad3 with the CXCR4 promoter. TGFβ/Smad3 treatment also markedly enhanced SDF‐1α‐induced ERK1/2 phosphorylation as well as SMC migration in a CXCR4‐dependent manner. Adenoviral expression of Smad3 in balloon‐injured rat carotid arteries increased local CXCR4 levels and enhanced IH, whereas SMC‐specific depletion of CXCR4 in the wire‐injured mouse femoral arterial wall produced a 60% reduction in IH. Our results provide the first evidence that upregulation of TGFβ/Smad3 in injured arteries induces local SMC CXCR4 expression and cell migration, and consequently IH. The Smad3/CXCR4 pathway may provide a potential target for therapeutic interventions to prevent restenosis. Stem Cells 2016;34:2744–2757
doi:10.1002/stem.2442
PMCID: PMC5113668  PMID: 27340942
CXCR4/SDF‐1α; TGFβ/Smad3; smooth muscle cell migration; smooth muscle cell specific CXCR4 knockout; intimal hyperplasia
15.  Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis 
Stem cells (Dayton, Ohio)  2015;33(6):1850-1862.
Heparanase plays important roles in tumor angiogenesis. Our previous study demonstrated that hypoxic preconditioning (HPC) enhanced the angiogenic and therapeutic effects of mesenchymal stem cells (MSCs), effects that were paralleled by enhanced heparanase expression. This study was designed to elucidate the role of heparanase in the improved therapeutic properties of HPC-MSCs and to explore underlying mechanisms using an ischemic rat hind limb model. MSCs transfected with heparanase (MSChpa) or empty vector (MSCnull) were delivered by intramuscular injections to ischemic hind limbs. Hind limbs that received MSChpa recovered blood flow more rapidly at 7 days and acquired higher capillary density at 14 days compared with MSCnull. Conditioned medium from MSChpa increased endothelial cell migration and promoted greater tube formation relative to that from the MSCnull groups. Vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1) and its downstream signaling pathway (p38MAPK/HSP27) were significantly increased in human umbilical vein endothelial cells (HUVECs) after treatment with MSChpa conditioned medium. Each of these responses was decreased by cocultured with MSChpa-KD conditioned medium. MSChpa conditioned medium activated hypoxia-inducible factor-2α (HIF-2α) and increased in parallel the transcript level of Flk-1 as determined by chromatin immunoprecipitation-PCR and luciferase assays. Analyses of integrin expression revealed an important role for integrin β1 in the regulation of HIF-2α. All angiogenic effects of MSChpa conditioned medium were abolished by knockdown of integrin β1, HIF-2α, and Flk-1 in HUVECs with selective shRNAs. These findings identify heparanse as a key regulator of angiogenesis by MSCs. We propose a novel pathway wherein heparanse sequentially activates integrin β1, HIF-2α, Flk-1, and p38MAPK/HSP27 with corresponding enhancement of angiogenesis.
doi:10.1002/stem.1995
PMCID: PMC5108061  PMID: 25754303
Heparanase; Angiogenesis; Mesenchymal stem cell
16.  Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells 
Stem cells (Dayton, Ohio)  2016;34(8):2079-2089.
Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs.
doi:10.1002/stem.2427
PMCID: PMC5097445  PMID: 27331788
Human embryonic stem cells; Human induced pluripotent stem cells; Yellow fluorescent protein; SMAD pathway; Fibrinogen; α9β1; RUNX2; Osteoblast
17.  Leptin Signaling Is Required for Augmented Therapeutic Properties of Mesenchymal Stem Cells Conferred by Hypoxia Preconditioning 
Stem cells (Dayton, Ohio)  2014;32(10):2702-2713.
Hypoxia preconditioning enhances the therapeutic effect of mesenchymal stem cells (MSCs). However, the mechanism underlying hypoxia-induced augmentation of the protective effect of MSCs on myocardial infarction (MI) is poorly understood. We show that hypoxia-enhanced survival, mobility, and protection of cocultured cardiomyocytes were paralleled by increased expression of leptin and cell surface receptor CXCR4. The enhanced activities were abolished by either knockdown of leptin with a selective shRNA or by genetic deficiency of leptin or its receptor in MSCs derived, respectively, from ob/ob or db/db mice. To characterize the role of leptin in the regulation of MSC functions by hypoxia and its possible contribution to enhanced therapeutic efficacy, cell therapy using MSCs derived from wild-type, ob/ob, or db/db mice was implemented in mouse models of acute MI. Augmented protection by hypoxia pretreatment was only seen with MSCs from wild-type mice. Parameters that were differentially affected by hypoxia pretreatment included MSC engraftment, c-Kit+ cell recruitment to the infarct, vascular density, infarct size, and long-term contractile function. These data show that leptin signaling is an early and essential step for the enhanced survival, chemotaxis, and therapeutic properties of MSCs conferred by preculture under hypoxia. Leptin may play a physiological role in priming MSCs resident in the bone marrow endosteum for optimal response to systemic signaling molecules and subsequent tissue repair.
doi:10.1002/stem.1784
PMCID: PMC5096299  PMID: 24989835
Mesenchymal stem cells; Hypoxic preconditioning; Leptin; Myocardial infarction; CXCR4
18.  MiR-211/STAT5A Signaling Modulates Migration of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy 
Stem cells (Dayton, Ohio)  2016;34(7):1846-1858.
Our previous study showed that the therapeutic effects of mesenchymal stem cells (MSCs) transplantation were improved by enhancing migration. MicroRNA-211 (miR-211) can modulate the migratory properties of some cell types by mechanisms that are not fully understood. This study was designed to investigate a possible role for miR-211 in MSC migration, and whether genetic manipulation of miR-211 in MSCs could be used to enhance its beneficial effects of cell transplantation. Transwell assays confirmed that MSCs migration of was significantly impaired by miR-211 knockdown but enhanced by miR-211 overexpression. MiR-211 overexpressing MSCs also exhibited significantly increased cell engraftment in the peri-infarct areas of female rat hearts 2 days after intravenous transplantation of male MSCs as shown by GFP tracking and SYR gene quantification. This conferred a significant decrease in infarct size and improved cardiac performance. By using a loss or gain of gene function approach, we demonstrated that miR-211 targeted STAT5A to modulate MSCs migration, possibly by interacting with MAPK signaling. Furthermore, the beneficial effects of miR-211 overexpression in MSCs were abolished by simultaneous overexpression of STAT5A whereas the negative effects of miR-211 silencing on MSC migration were rescued by simultaneous downregulation of STAT5A. Finally, using ChIP-PCR and luciferase assays, we provide novel evidence that STAT3 can directly bind to promoter elements that activate miR-211 expression. STAT3/miR-211/STAT5A signaling plays a key role in MSCs migration. Intravenous infusion of genetically modified miR-211 overexpressing MSCs conveys enhanced protection from adverse post-MI remodeling compared with unmodified MSCs.
doi:10.1002/stem.2391
PMCID: PMC5096301  PMID: 27145179
Mesenchymal stem cells; MicroRNA-211; STAT5A; Migration; Retention; Myocardial infarction
19.  Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine 
Stem cells (Dayton, Ohio)  2015;33(11):3165-3173.
Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past two decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology and in regenerative medicine.
doi:10.1002/stem.2079
PMCID: PMC4618071  PMID: 26086534
embryonic stem cells; innate immunity; antiviral response; type I interferons; regenerative medicine
20.  Wnt/β-catenin-Responsive Cells in Prostatic Development and Regeneration 
Stem cells (Dayton, Ohio)  2015;33(11):3356-3367.
The precise role of Wnt/β-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of β-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of β-catenin or expression of stabilized β-catenin in these Axin2-expressing cells results in abnormal development and oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/β-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/β-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis.
doi:10.1002/stem.2096
PMCID: PMC4618081  PMID: 26220362
Wnt Signaling; β-catenin; Axin2; Prostatic progenitor cells
21.  Fanconi anemia mesenchymal stromal cells-derived glycerophospholipids skew hematopoietic stem cell differentiation through Toll-like receptor signaling 
Stem cells (Dayton, Ohio)  2015;33(11):3382-3396.
Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids and their endogenous inhibitor, 5-(Tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells (HSPCs). We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (1) limiting-dilution CAFC assay revealed that TOFA significantly increased cobblestone colonies in Fanca−/− or Fancd2−/− co-cultures compared to untreated co-cultures. (2) Competitive repopulating assay using output cells collected from co-cultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca−/− or Fancd2−/− co-cultures. Further, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting Glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation.
doi:10.1002/stem.2100
PMCID: PMC4618082  PMID: 26212365
22.  The Theory and Practice of Lineage Tracing 
Stem cells (Dayton, Ohio)  2015;33(11):3197-3204.
Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology, and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed.
doi:10.1002/stem.2123
PMCID: PMC4618107  PMID: 26284340
23.  Density-Dependent Metabolic Heterogeneity in Human Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2015;33(11):3368-3381.
Human mesenchymal stem cells (hMSCs) are intrinsically heterogeneous and comprise subpopulations that differ in their proliferation, multi-potency, and functional properties, which are commonly demonstrated by culturing hMSCs at different plating densities. The objective of this study was to investigate the metabolic profiles of different subpopulations of hMSC by testing the hypothesis that the clonogenic hMSC subpopulation, which is selectively enriched in clonal density (CD) and low density (LD) culture (10 and 100 cells per square centimeter, respectively), possesses a metabolic phenotype that differs from that of hMSC in medium- or high-density (MD: 1,000 and HD: 3,000 cells per square centimeter, respectively). Cells at CD and LD conditions exhibited elevated expression of CD146 and colony forming unit-fibroblast compared with cells at MD- or HD. Global metabolic profiles revealed by gas chromatography-mass spectrometry of cell extracts showed clear distinction between LD and HD cultures, and density-dependent differences in coupling of glycolysis to the TCA cycle. Metabolic inhibitors revealed density-dependent differences in glycolysis versus oxidative phosphorylation (OXPHOS) for ATP generation, in glutamine metabolism, in the dependence on the pentose phosphate pathway for maintaining cellular redox state, and sensitivity to exogenous reactive oxygen species. We also show that active OXPHOS is not required for proliferation in LD culture but that OXPHOS activity increases senescence in HD culture. Together, the results revealed heterogeneity in hMSC culture exists at the level of primary metabolism. The unique metabolic characteristics of the clonogenic subpopulation suggest a novel approach for optimizing in vitro expansion of hMSCs.
doi:10.1002/stem.2097
PMCID: PMC4757908  PMID: 26274841
Mesenchymal stem cells; Cellular proliferation; Hypoxia; Cellular therapy
24.  Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy 
Stem cells (Dayton, Ohio)  2015;33(5):1532-1542.
Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34+/CD38− cells, comprising ~1%–3% of all CD34+ cells, were isolated from healthy cord blood CD34+ cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-βAS3-FB). Isolated CD34+/CD38− cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34+ cells and required up to 40-fold less vector for transduction compared to bulk CD34+ preparations containing an equivalent number of CD34+/CD38− cells. Transduction of isolated CD34+/CD38− cells was comparable to CD34+ cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34+/38− cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34+/CD38− cells or unfractionated CD34+ cells. In vivo studies showed equivalent engraftment of transduced CD34+/CD38− cells when transplanted in competition with 100-fold more CD34+/CD38+ cells. This work provides initial evidence for the beneficial effects from isolating human CD34+/CD38− cells to use significantly less vector and potentially improve transduction for HSC gene therapy.
doi:10.1002/stem.1957
PMCID: PMC5074681  PMID: 25588820
Hematopoietic stem cells; Gene therapy; Lentiviral vector; Stem cell enrichment
25.  Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases 
Stem cells (Dayton, Ohio)  2015;33(10):2985-2994.
The treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer has been revolutionized by trastuzumab. However, longer survival of these patients now predisposes them to forming HER2 positive brain metastases, as the therapeutic antibodies cannot cross the blood brain barrier. The current oncologic repertoire does not offer a rational, non-toxic targeted therapy for brain metastases. In this study, we utilized an established human neural stem cell line, HB1.F3 NSCs, and generated a stable pool of cells secreting a high amount of functional full-length anti-HER2 antibody, equivalent to trastuzumab. Anti-HER2Ab secreted by the NSCs (HER2Ab-NSCs) specifically binds to HER2 overexpressing human breast cancer cells and inhibits PI3K-Akt signaling. This translates to HER2Ab-NSC inhibition of breast cancer cell growth in vitro. Pre-clinical in vivo experiments using HER2Ab overexpressing NSCs in a breast cancer brain metastases (BCBM) mouse model demonstrate that intracranial injection of HER2Ab-NSCs significantly improves survival. In effect, these NSCs provide tumor localized production of HER2Ab, minimizing any potential off-target side effects. Our results establish HER2Ab-NSCs as a novel, non-toxic and rational therapeutic approach for the successful treatment of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation.
Graphical Abstract
doi:10.1002/stem.2109
PMCID: PMC4578995  PMID: 26260958
Neural stem cells; Breast cancer brain metastasis; Human epidermal growth factor receptor 2; HER2 overexpressing breast cancer; Trastuzumab; Blood brain barrier; Monoclonal antibody therapy

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