Browsing by Subject "Stem Cells"
Now showing 1 - 20 of 29
Results Per Page
Sort Options
Item Open Access A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.(G3 (Bethesda, Md.), 2016-08) Ables, Elizabeth T; Hwang, Grace H; Finger, Danielle S; Hinnant, Taylor D; Drummond-Barbosa, DanielaMultiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.Item Open Access Age-related changes in the cellular composition and epithelial organization of the mouse trachea.(PloS one, 2014-01) Wansleeben, Carolien; Bowie, Emily; Hotten, Danielle F; Yu, Yen-Rei A; Hogan, Brigid LMWe report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.Item Open Access Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.(Dis Model Mech, 2010-09) Rock, Jason R; Randell, Scott H; Hogan, Brigid LMThe small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.Item Open Access Antibody-Armed Platelets for the Regenerative Targeting of Endogenous Stem Cells.(Nano letters, 2019-03) Shen, Deliang; Li, Zhenhua; Hu, Shiqi; Huang, Ke; Su, Teng; Liang, Hongxia; Liu, Feiran; Cheng, KeStem cell therapies have shown promise in treating acute and chronic ischemic heart disease. However, current therapies are limited by the low retention and poor integration of injected cells in the injured tissue. Taking advantage of the natural infarct-homing ability of platelets, we engineered CD34 antibody-linked platelets (P-CD34) to capture circulating CD34-positive endogenous stem cells and direct them to the injured heart. In vitro, P-CD34 could bind to damaged aortas and capture endogenous stem cells in whole blood. In a mouse model of acute myocardial infarction, P-CD34 accumulated in the injured heart after intravenous administration, leading to a concentration of endogenous CD34 stem cells in the injured heart for effective heart repair. This represents a new technology for endogenous stem cell therapy.Item Open Access Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.(Development, 2010-09) Hutson, MR; Zeng, XL; Kim, AJ; Antoon, E; Harward, S; Kirby, MLDuring heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.Item Open Access Cardiac Stem Cell Patch Integrated with Microengineered Blood Vessels Promotes Cardiomyocyte Proliferation and Neovascularization after Acute Myocardial Infarction.(ACS applied materials & interfaces, 2018-10) Su, Teng; Huang, Ke; Daniele, Michael A; Hensley, Michael Taylor; Young, Ashlyn T; Tang, Junnan; Allen, Tyler A; Vandergriff, Adam C; Erb, Patrick D; Ligler, Frances S; Cheng, KeCardiac stem cell (CSC) therapy has shown preclinical and clinical evidence for ischemic heart repair but is limited by low cellular engraftment and survival after transplantation. Previous versions of the cardiac patch strategy improve stem cell engraftment and encourage repair of cardiac tissue. However, cardiac patches that can enhance cardiomyogenesis and angiogenesis at the injured site remain elusive. Therapies that target cardiomyocyte proliferation and new blood vessel formation hold great potential for the protection against acute myocardial infarction (MI). Here, we report a new strategy for creating a vascularized cardiac patch in a facile and modular fashion by leveraging microfluidic hydrodynamic focusing to construct the biomimetic microvessels (BMVs) that include human umbilical vein endothelial cells (HUVECs) lining the luminal surface and then encapsulating the BMVs in a fibrin gel spiked with human CSCs. We show that the endothelialized BMVs mimicked the natural architecture and function of capillaries and that the resultant vascularized cardiac patch (BMV-CSC patch) exhibited equivalent release of paracrine factors compared to those of coculture of genuine human CSCs and HUVECs after 7 days of in vitro culture. In a rat model of acute MI, the BMV-CSC patch therapy induced profound mitotic activities of cardiomyocytes in the peri-infarct region 4 weeks post-treatment. A significant increase in myocardial capillary density was noted in the infarcted hearts that received BMV-CSC patch treatment compared to the infarcted hearts treated with conventional CSC patches. The striking therapeutic benefits and the fast and facile fabrication of the BMV-CSC patch make it promising for practical applications. Our findings suggest that the BMV-CSC patch strategy may open up new possibilities for the treatment of ischemic heart injury.Item Open Access Defined conditions for long-term expansion of murine and human alveolar epithelial stem cells in three-dimensional cultures.(STAR protocols, 2022-06-10) Konishi, Satoshi; Tata, Aleksandra; Tata, Purushothama RaoAlveolar type 2 cells (AT2s) serve as stem cells of the alveoli and restore cell numbers after injury. Here, we describe a detailed protocol for the isolation, purification, and culture of murine and human AT2s. We have developed chemically defined and stroma-free culture conditions that enable expansion and maintenance of AT2s. The culture conditions are scalable and compatible with high-throughput chemical and genetic screenings and can potentially be used to generate large AT2 numbers for cell-based therapies. For complete details on the use and execution of this protocol, please refer to Katsura et al. (2020).Item Open Access Designing topographically textured microparticles for induction and modulation of osteogenesis in mesenchymal stem cell engineering.(Biomaterials, 2021-01) Amer, Mahetab H; Alvarez-Paino, Marta; McLaren, Jane; Pappalardo, Francesco; Trujillo, Sara; Wong, Jing Qian; Shrestha, Sumana; Abdelrazig, Salah; Stevens, Lee A; Lee, Jong Bong; Kim, Dong-Hyun; González-García, Cristina; Needham, David; Salmerón-Sánchez, Manuel; Shakesheff, Kevin M; Alexander, Morgan R; Alexander, Cameron; Rose, Felicity RajMesenchymal stem cells are the focus of intense research in bone development and regeneration. The potential of microparticles as modulating moieties of osteogenic response by utilizing their architectural features is demonstrated herein. Topographically textured microparticles of varying microscale features are produced by exploiting phase-separation of a readily soluble sacrificial component from polylactic acid. The influence of varying topographical features on primary human mesenchymal stem cell attachment, proliferation and markers of osteogenesis is investigated. In the absence of osteoinductive supplements, cells cultured on textured microparticles exhibit notably increased expression of osteogenic markers relative to conventional smooth microparticles. They also exhibit varying morphological, attachment and proliferation responses. Significantly altered gene expression and metabolic profiles are observed, with varying histological characteristics in vivo. This study highlights how tailoring topographical design offers cell-instructive 3D microenvironments which allow manipulation of stem cell fate by eliciting the desired downstream response without use of exogenous osteoinductive factors.Item Open Access Distal-Less Homeobox 5 Is a Therapeutic Target for Attenuating Hypertrophy and Apoptosis of Mesenchymal Progenitor Cells.(International journal of molecular sciences, 2020-07) Twomey-Kozak, John; Desai, Salomi; Liu, Wenguang; Li, Neill Y; Lemme, Nicholas; Chen, Qian; Owens, Brett D; Jayasuriya, Chathuraka TChondrocyte hypertrophy is a hallmark of osteoarthritis (OA) pathology. In the present study, we elucidated the mechanism underlying the relationship between the hypertrophy/apoptotic phenotype and OA pathogenesis in bone marrow-derived mesenchymal stem cells (BM-MSCs) via gene targeting of distal-less homeobox 5 (DLX5). Our primary objectives were (1) to determine whether DLX5 is a predictive biomarker of cellular hypertrophy in human osteoarthritic tissues; (2) To determine whether modulating DLX5 activity can regulate cell hypertrophy in mesenchymal stem/progenitor cells from marrow and cartilage. Whole transcriptome sequencing was performed to identify differences in the RNA expression profile between human-cartilage-derived mesenchymal progenitors (C-PCs) and bone-marrow-derived mesenchymal progenitors (BM-MSCs). Ingenuity Pathway Analysis (IPA) software was used to compare molecular pathways known to regulate hypertrophic terminal cell differentiation. RT-qPCR was used to measure DLX5 and hypertrophy marker COL10 in healthy human chondrocytes and OA chondrocytes. DLX5 was knocked down or overexpressed in BM-MSCs and C-PCs and RT-qPCR were used to measure the expression of hypertrophy/terminal differentiation markers following DLX5 modulation. Apoptotic cell activity was characterized by immunostaining for cleaved caspase 3/7. We demonstrate that DLX5 and downstream hypertrophy markers were significantly upregulated in BM-MSCs, relative to C-PCs. DLX5 and COL10 were also significantly upregulated in cells from OA knee joint tissues, relative to normal non-arthritic joint tissues. Knocking down DLX5 in BM-MSCs inhibited cell hypertrophy and apoptotic activity without attenuating their chondrogenic potential. Overexpression of DLX5 in C-PCs stimulated hypertrophy markers and increased apoptotic cell activity. Modulating DLX5 activity regulates cell hypertrophy and apoptosis in BM-MSCs and C-PCs. These findings suggest that DLX5 is a biomarker of OA changes in human knee joint tissues and confirms the DLX5 mechanism contributes to hypertrophy and apoptosis in BM-MSCs.Item Open Access Enhanced In Vivo Delivery of Stem Cells using Microporous Annealed Particle Scaffolds.(Small (Weinheim an der Bergstrasse, Germany), 2019-09) Koh, Jaekyung; Griffin, Donald R; Archang, Maani M; Feng, An-Chieh; Horn, Thomas; Margolis, Michael; Zalazar, David; Segura, Tatiana; Scumpia, Philip O; Di Carlo, DinoDelivery to the proper tissue compartment is a major obstacle hampering the potential of cellular therapeutics for medical conditions. Delivery of cells within biomaterials may improve localization, but traditional and newer void-forming hydrogels must be made in advance with cells being added into the scaffold during the manufacturing process. Injectable, in situ cross-linking microporous scaffolds are recently developed that demonstrate a remarkable ability to provide a matrix for cellular proliferation and growth in vitro in three dimensions. The ability of these scaffolds to deliver cells in vivo is currently unknown. Herein, it is shown that mesenchymal stem cells (MSCs) can be co-injected locally with microparticle scaffolds assembled in situ immediately following injection. MSC delivery within a microporous scaffold enhances MSC retention subcutaneously when compared to cell delivery alone or delivery within traditional in situ cross-linked nanoporous hydrogels. After two weeks, endothelial cells forming blood vessels are recruited to the scaffold and cells retaining the MSC marker CD29 remain viable within the scaffold. These findings highlight the utility of this approach in achieving localized delivery of stem cells through an injectable porous matrix while limiting obstacles of introducing cells within the scaffold manufacturing process.Item Open Access Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer.(PLoS One, 2011) Philips, GM; Chan, IS; Swiderska, M; Schroder, VT; Guy, C; Karaca, GF; Moylan, C; Venkatraman, T; Feuerlein, S; Syn, WK; Jung, Y; Witek, RP; Choi, S; Michelotti, GA; Rangwala, F; Merkle, E; Lascola, C; Diehl, AMOBJECTIVE: Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50-60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2(-/-) mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC. METHODS: Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2(-/-) mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging. RESULTS: Unlike controls, Mdr2(-/-) mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality. CONCLUSIONS: Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.Item Open Access Human distal lung maps and lineage hierarchies reveal a bipotent progenitor.(Nature, 2022-04) Kadur Lakshminarasimha Murthy, Preetish; Sontake, Vishwaraj; Tata, Aleksandra; Kobayashi, Yoshihiko; Macadlo, Lauren; Okuda, Kenichi; Conchola, Ansley S; Nakano, Satoko; Gregory, Simon; Miller, Lisa A; Spence, Jason R; Engelhardt, John F; Boucher, Richard C; Rock, Jason R; Randell, Scott H; Tata, Purushothama RaoMapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.Item Open Access Identification of select glucocorticoids as Smoothened agonists: potential utility for regenerative medicine.(Proc Natl Acad Sci U S A, 2010-05-18) Wang, Jiangbo; Lu, Jiuyi; Bond, Michael C; Chen, Minyong; Ren, Xiu-Rong; Lyerly, H Kim; Barak, Larry S; Chen, WeiRegenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a beta-arrestin2-GFP reporter, we identified four FDA-approved drugs, halcinonide, fluticasone, clobetasol, and fluocinonide, as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo, promote Smo internalization, activate Gli, and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide, fluticasone, clobetasol, and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses.Item Open Access Identifying Treatments for Taste and Smell Disorders: Gaps and Opportunities.(Chemical senses, 2020-10) Mainland, Joel D; Barlow, Linda A; Munger, Steven D; Millar, Sarah E; Vergara, M Natalia; Jiang, Peihua; Schwob, James E; Goldstein, Bradley J; Boye, Shannon E; Martens, Jeffrey R; Leopold, Donald A; Bartoshuk, Linda M; Doty, Richard L; Hummel, Thomas; Pinto, Jayant M; Trimmer, Casey; Kelly, Christine; Pribitkin, Edmund A; Reed, Danielle RThe chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.Item Open Access Interstitial engraftment of adipose-derived stem cells into an acellular dermal matrix results in improved inward angiogenesis and tissue incorporation.(J Biomed Mater Res A, 2013-10) Komatsu, Issei; Yang, Jun; Zhang, Ying; Levin, L Scott; Erdmann, D; Klitzman, Bruce; Hollenbeck, Scott TAcellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 10(5) cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs.Item Open Access Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood.(Cytotherapy, 2011-07) Tracy, Elisabeth T; Zhang, Claire Y; Gentry, Tracy; Shoulars, Kevin W; Kurtzberg, JoanneBackground aims
Oligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCB.Methods
We thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination.Results
The average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.Conclusions
Our results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.Item Open Access Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.(Tissue Eng Part A, 2010-07) Little, Dianne; Guilak, Farshid; Ruch, David SHuman adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (pItem Open Access Multi-site evaluation of the BD Stem Cell Enumeration Kit for CD34(+) cell enumeration on the BD FACSCanto II and BD FACSCalibur flow cytometers.(Cytotherapy, 2014-11) Preti, Robert A; Chan, Wai Shun; Kurtzberg, Joanne; Dornsife, Ronna E; Wallace, Paul K; Furlage, Rosemary; Lin, Anna; Omana-Zapata, Imelda; Bonig, Halvard; Tonn, TorstenBackground aims
Evaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34(+) enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France).Methods
Leftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose-solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis.Results
The mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (-2.81 to 4.31 ±7.1) and BD FACSCalibur (-2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+) populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+). Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R(2) > 0.92 for both investigational methods.Discussion
In conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34(+), percentage of viable CD34(+) in CD45(+) and absolute viable CD45(+) populations.Item Open Access Patient-derived endothelial progenitor cells improve vascular graft patency in a rodent model.(Acta Biomater, 2012-01) Stroncek, JD; Ren, LC; Klitzman, B; Reichert, WMLate outgrowth endothelial progenitor cells (EPCs) derived from the peripheral blood of patients with significant coronary artery disease were sodded into the lumens of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts. Grafts (1mm inner diameter) were denucleated and sodded either with native EPCs or with EPCs transfected with an adenoviral vector containing the gene for human thrombomodulin (EPC+AdTM). EPC+AdTM was shown to increase the in vitro rate of graft activated protein C (APC) production 4-fold over grafts sodded with untransfected EPCs (p<0.05). Unsodded control and EPC-sodded and EPC+AdTM-sodded grafts were implanted bilaterally into the femoral arteries of athymic rats for 7 or 28 days. Unsodded control grafts, both with and without denucleation treatment, each exhibited 7 day patency rates of 25%. Unsodded grafts showed extensive thrombosis and were not tested for patency over 28 days. In contrast, grafts sodded with untransfected EPCs or EPC+AdTM both had 7 day patency rates of 88-89% and 28 day patency rates of 75-88%. Intimal hyperplasia was observed near both the proximal and distal anastomoses in all sodded graft conditions but did not appear to be the primary occlusive failure event. This in vivo study suggests autologous EPCs derived from the peripheral blood of patients with coronary artery disease may improve the performance of synthetic vascular grafts, although no differences were observed between untransfected EPCs and TM transfected EPCs.Item Open Access Pretargeting and Bioorthogonal Click Chemistry-Mediated Endogenous Stem Cell Homing for Heart Repair.(ACS nano, 2018-12) Li, Zhenhua; Shen, Deliang; Hu, Shiqi; Su, Teng; Huang, Ke; Liu, Feiran; Hou, Lei; Cheng, KeStem cell therapy is one of the promising strategies for the treatment of ischemic heart disease. However, the clinical application of stem cells transplantation is limited by low cell engraftment in the infarcted myocardium. Taking advantage of pretargeting and bioorthogonal chemistry, we engineered a pretargeting and bioorthogonal chemistry (PTBC) system to capture endogenous circulating stem cells and target them to the injured heart for effective repair. Two bioorthogonal antibodies were i.v. administrated with a pretargeting interval (48 h). Through bioorthogonal click reaction, the two antibodies are linked in vivo, engaging endogenous stem cells with circulating platelets. As a result, the platelets redirect the stem cells to the injured heart. In vitro and in vivo studies demonstrated that bioorthogonal click reaction was able to induce the conjugation of platelets and endothelial progenitor cells (EPCs) and enhance the binding of EPCs to collagen and injured blood vessels. More importantly, in a mouse model of acute myocardial infarction, the in vivo results of cardiac function, heart morphometry, and immunohistochemistry assessment all confirmed effective heart repair by the PTBC system.