34 results on '"Mack, David L."'
Search Results
2. Disease-in-a-Dish.
- Author
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Mack, David L., Xuan Guan, Wagoner, Ashley, Walker, Stephen J., and Childers, Martin K.
- Subjects
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AUTISM , *BIOMEDICAL engineering , *DUCHENNE muscular dystrophy , *HEART diseases , *NEUROLOGICAL disorders , *REGENERATION (Biology) , *REHABILITATION , *STEM cells , *TECHNOLOGY - Abstract
Advances in regenerative medicine technologies will lead to dramatic changes in how patients in rehabilitation medicine clinics are treated in the upcoming decades. The multidisciplinary field of regenerative medicine is developing new tools for disease modeling and drug discovery based on induced pluripotent stem cells. This approach capitalizes on the idea of personalized medicine by using the patient's own cells to discover new drugs, increasing the likelihood of a favorable outcome. The search for compounds that can correct disease defects in the culture dish is a conceptual departure from how drug screens were done in the past. This system proposes a closed loop from sample collection from the diseased patient, to in vitro disease model, to drug discovery and Food and Drug Administration approval, to delivering that drug back to the same patient. Here, recent progress in patient-specific induced pluripotent stem cell derivation, directed differentiation toward diseased cell types, and how those cells can be used for high-throughput drug screens are reviewed. Given that restoration of normal function is a driving force in rehabilitation medicine, the authors believe that this drug discovery platform focusing on phenotypic rescue will become a key contributor to therapeutic compounds in regenerative rehabilitation. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
3. Genetic Modification of Primate Amniotic Fluid-Derived Stem Cells Produces Pancreatic Progenitor Cells in vitro.
- Author
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Zhou, Yu, Mack, David L., Williams, J. Koudy, Mirmalek-Sani, Sayed-Hadi, Moorefield, Emily, Chun, So-Young, Wang, Jun, Lorenzetti, Diego, Furth, Mark, atala, anthony, and Soker, Shay
- Subjects
- *
INSULIN therapy , *DIABETES complications , *VASCULAR diseases , *PERIPHERAL vascular diseases , *NEUROPATHY , *DIABETIC retinopathy , *KIDNEY failure - Abstract
Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for β-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a β-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
4. The mammary microenvironment alters the differentiation repertoire of neural stem cells.
- Author
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Booth, Brian W., Mack, David L., AndroutseIIis-Theotokis, Andreas, McKay, Ronald D. G., Boulanger, Corinne A., and Smith, Gilbert H.
- Subjects
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STEM cells , *MATERIAL plasticity , *EPITHELIAL cells , *REGENERATION (Biology) , *ECOLOGICAL niche , *MAMMARY glands , *TRANSPLANTATION of cell nuclei - Abstract
A fundamental issue in stem cell biology is whether adult somatic stem cells are capable of accessing alternate tissue sites and continue functioning as stem cells in the new microenvironment. To address this issue relative to neurogenic stem cells in the mouse mammary gland microenvironment, we mixed wild-type mam- mary epithelial cells (MECs) with bona fide neural stem cells (NSCs) isolated from WAP-Cre/Rosa26R mice and inoculated them into cleared fat pads of immunocompromised females. Hosts were bred 6-8 weeks later and examined postinvolution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination, and constitutive expression of LacZ. The NSCs and their progeny contributed to mammary epithelial growth during ductal morphogenesis, and the Rosa26-LacZ reporter gene was activated by WAP-Cre expression during pregnancy. Some NSC- derived LacZ[sup+] cells expressed mammary-specific functions, includ- ing milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSC5 and their progeny enter mammary epithelium- specific niches and adopt the function of similarly endowed mam- mary cells. This result supports the conclusion that tissue-specific signals emanating from the stroma and from the differentiated somatic cells of the mouse mammary gland can redirect the NSCs to produce cellular progeny committed to MEC fates. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
5. Interaction with the mammary microenvironment redirects spermatogenic cell fate in vivo.
- Author
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Boulanger, Corinne A., Mack, David L., Booth, Brian W., and Smith, Gilbert H.
- Subjects
- *
EPITHELIAL cells , *CELL proliferation , *TRANSPLANTATION of cell nuclei , *MAMMARY glands , *CELL determination , *PREGNANCY - Abstract
Previously, we characterized a parity-induced mammary epithelial cell population that possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of β-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through Cre-lox recombination during pregnancy. We used this experimental model to determine whether testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation, and involution. Adult testicular cells, isolated from seminiferous tubules, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. The host mice were bred 6-8 weeks later and examined 20-30 days postinvolution. This approach allowed for the growth of mammary tissue from the injected cells and transient activation of the whey acidic protein promoter-Cre gene during pregnancy and lactation, leading to Cre-lox recombination and constitutive expression of LacZ from its promoter. Here we show that cells from adult seminiferous tubules interact with mammary epithelial cells during regeneration of the gland. They adopt mammary epithelial progenitor cell properties, including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells. Our results provide evidence for the ascendancy of the tissue microenvironment over the intrinsic nature of cells from an alternative adult tissue. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
6. Down-regulation of the myeloid homeobox protein Hex is essential for normal T-cell development.
- Author
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Mack, David L., Leibowitz, David S., Cooper, Scott, Ramsey, Heather, Broxmeyer, Hal E., and Hromas, Robert
- Subjects
- *
HOMEOBOX genes , *MYELOID metaplasia - Abstract
The haematopoietic homeobox gene Hex (also called Prh) is expressed in myeloid cells and B cells but not T cells. To investigate whether Hex levels might play a role in myeloid versus T-cell development, two types of transgenic mouse lines were constructed, each with ectopic expression of Hex in T cells (CD11a/Hex and Lck/Hex). Both these types of transgenic mouse had the same defects in T-cell maturation, indicating that proper T-cell development may be dependent not just on the up-regulation of lymphoid-specific transcriptional regulators but also on the co-ordinated down-regulation of myeloid-specific transcriptional regulators such as Hex. In addition, Hex over-expression significantly increased myeloid progenitor cycling, which may explain its role in retrovirally induced murine leukaemia. [ABSTRACT FROM AUTHOR]
- Published
- 2002
- Full Text
- View/download PDF
7. Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.
- Author
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Soah Lee, Vander Roest, Alison S., Blair, Cheavar A., Kao, Kerry, Bremner, Samantha B., Childers, Matthew C., Pathak, Divya, Heinrich, Paul, Lee, Daniel, Chirikian, Orlando, Mohran, Saffie E., Roberts, Brock, Smith, Jacqueline E., Jahng, James W., Paik, David T., Wu, Joseph C., Gunawardane, Ruwanthi N., Ruppel, Kathleen M., Mack, David L., and Pruitt, Beth L.
- Subjects
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HYPERTROPHIC cardiomyopathy , *CONTRACTILE proteins , *GENETIC variation , *RESPIRATION , *GENETIC mutation - Abstract
Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
8. The promyelocytic leukemia protein PML interacts with the proline-rich homeodomain protein PRH: a RING may link hematopoiesis and growth control.
- Author
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Topcu, Zeki, Mack, David L, Hromas, Robert A, and Borden, Katherine LB
- Subjects
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LEUKEMIA , *PROLINE , *HEMATOPOIESIS - Abstract
Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML is disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis. [ABSTRACT FROM AUTHOR]
- Published
- 1999
- Full Text
- View/download PDF
9. THE U.S. AND LIBYA: NORMALIZATION OF A STORMY RELATIONSHIP.
- Author
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Mack, David L.
- Subjects
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INTERNATIONAL relations , *PEACE treaties , *AMERICAN air bases , *MILITARY education ,LIBYA-United States relations - Abstract
The article discusses the international relations between Libya and the U.S. It offers a brief history on foreign policies of the two countries such as peace treaty, naval base, and agricultural settlement refute. It states that the U.S. has rendered services to Libya including rental fees in air bases and military trainings. It also cites the process of normalization of Libya-U.S. relations and plans of U.S. President Barack Obama's administration to expand bilateral relations with Tripoli.
- Published
- 2011
10. Arab-Israeli negotiations and U.S. interests in the Middle East: Second-term imperatives.
- Author
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Miller, Aaron David and Mack, David L.
- Subjects
- *
PEACE movements - Abstract
Investigates the viability of peace negotiations aimed at ending the Arab-Israeli conflict and its implication to the advancement of United States interests in the Middle East. Maturity of political conditions leading to conflict cessation; Issues other than the Arab-Israeli conflict interwoven with Gulf security; Crisis reshaping internal Israeli politics.
- Published
- 1997
- Full Text
- View/download PDF
11. LIBYA: AN ALTERNATIVE PARADIGM.
- Author
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Mack, David L.
- Subjects
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DETENTE , *TERRORISM , *NATIONAL security , *ECONOMIC sanctions - Abstract
The article discusses the mutual benefits U.S.-Libya detente. Detente, including intelligence exchanges on mutual terrorist threats, provides the national security needs of the two countries. It makes Libya safer from future military pressures and economic sanctions. Also, Libya and the U.S. gained strategically in their relations from the detente, however it needs more forward movement to prevent a relapse.
- Published
- 2005
12. REALITY CHECK IN IRAQ.
- Author
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Mack, David L.
- Subjects
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RECONSTRUCTION in the Iraq War, 2003-2011 ,FOREIGN relations of the United States, 2001-2009 ,IRAQI foreign relations, 1991- - Abstract
The article presents the author's views regarding the U.S. role of rebuilding Iraq in the post Iraq war. The author states that the U.S. should adopt greater realism in its Iraq policies to attain modest and achievable expectations for Iraq. He observes that the political change and reconstruction in Iraq are hard tasks for the U.S. The article also discusses the risk of conducting elections in Iraq.
- Published
- 2005
13. Avoiding Grave Harm.
- Author
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Mack, David L.
- Subjects
- *
TERRORISM ,FOREIGN relations of the United States ,IRAQI foreign relations - Abstract
Discusses the likely development of United States (U.S.) policy toward Iraq following the September 11, 2001 terrorist attacks on the U.S. Benefits of the reintegration of Iraq into the international community; Potential threat of weapons of mass destruction in the hands of terrorists; Implications of the war against terrorism for Iraq.
- Published
- 2002
14. Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.
- Author
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Changho Chun, Jung Hyun Lee, Bothwell, Mark, Nghiem, Paul, Smith, Alec S. T., and Mack, David L.
- Subjects
- *
MOTOR neuron diseases , *AMYOTROPHIC lateral sclerosis , *MOTOR neurons , *IMMUNE checkpoint proteins , *PROGRAMMED death-ligand 1 , *BIOMARKERS , *DNA-binding proteins - Abstract
Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferongamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
15. Creating stem cell‐derived neuromuscular junctions in vitro.
- Author
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Luttrell, Shawn M., Smith, Alec S. T., and Mack, David L.
- Abstract
Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non‐invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell‐derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ‐in‐a‐dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease‐causing mutations disrupt structure, signaling, and function. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
16. High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing.
- Author
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Smith, Alec ST, Luttrell, Shawn M, Dupont, Jean-Baptiste, Gray, Kevin, Lih, Daniel, Fleming, Jacob W, Cunningham, Nathan J, Jepson, Sofia, Hesson, Jennifer, Mathieu, Julie, Maves, Lisa, Berry, Bonnie J, Fisher, Elliot C, Sniadecki, Nathan J, Geisse, Nicholas A, and Mack, David L
- Subjects
- *
SKELETAL muscle , *INDUCED pluripotent stem cells , *HIGH throughput screening (Drug development) , *MUSCULAR sense - Abstract
Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
17. Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues.
- Author
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Bremner, Samantha B, Mandrycky, Christian J, Leonard, Andrea, Padgett, Ruby M, Levinson, Alan R, Rehn, Ethan S, Pioner, J Manuel, Sniadecki, Nathan J, and Mack, David L
- Subjects
- *
PLURIPOTENT stem cells , *HEART , *DYSTROPHIN , *CARDIAC contraction , *DUCHENNE muscular dystrophy , *DRUG discovery , *MYOCARDIUM - Abstract
Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
18. Gene therapy in monogenic congenital myopathies.
- Author
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Guan, Xuan, Goddard, Melissa A., Mack, David L., and Childers, Martin K.
- Subjects
- *
MUSCLE diseases , *CONGENITAL disorders , *MUSCULAR dystrophy , *GENETIC mutation , *LABORATORY mice , *GENE therapy , *THERAPEUTICS - Abstract
Current treatment options for patients with monogenetic congenital myopathies (MCM) ameliorate the symptoms of the disorder without resolving the underlying cause. However, gene therapies are being developed where the mutated or deficient gene target is replaced. Preclinical findings in animal models appear promising, as illustrated by gene replacement for X-linked myotubular myopathy (XLMTM) in canine and murine models. Prospective applications and approaches to gene replacement therapy, using these disorders as examples, are discussed in this review. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
19. Gene Therapy for Inherited Muscle Diseases.
- Author
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Braun, Robynne, Zejing Wang, Mack, David L., and Childers, Martin K.
- Subjects
- *
GENE therapy , *MUSCLE diseases , *MUSCULAR dystrophy , *REHABILITATION - Abstract
The development of clinical vectors to correct genetic mutations that cause inherited myopathies and related disorders of skeletal muscle is advancing at an impressive rate. Adeno-associated virus vectors are attractive for clinical use because (1) adeno-associated viruses do not cause human disease and (2) these vectors are able to persist for years. New vectors are now becoming available as gene therapy delivery tools, and recent preclinical experiments have demonstrated the feasibility, safety, and efficacy of gene therapy with adeno-associated virus for long-term correction of muscle pathology and weakness in myotubularin-deficient canine and murine disease models. In this review, recent advances in the application of gene therapies to treat inherited muscle disorders are presented, including Duchenne muscular dystrophy and x-linked myotubular myopathy. Potential areas for therapeutic synergies between rehabilitation medicine and genetics are also discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
20. High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing.
- Author
-
Smith, Alec S. T., Luttrell, Shawn M., Dupont, Jean-Baptiste, Gray, Kevin, Lih, Daniel, Fleming, Jacob W., Cunningham, Nathan J., Jepson, Sofia, Hesson, Jennifer, Mathieu, Julie, Maves, Lisa, Berry, Bonnie J., Fisher, Elliot C., Sniadecki, Nathan J., Geisse, Nicholas A., and Mack, David L.
- Subjects
- *
SKELETAL muscle , *INDUCED pluripotent stem cells , *HIGH throughput screening (Drug development) , *MUSCULAR sense - Abstract
Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
21. Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues.
- Author
-
Bremner, Samantha B., Mandrycky, Christian J., Leonard, Andrea, Padgett, Ruby M., Levinson, Alan R., Rehn, Ethan S., Pioner, J. Manuel, Sniadecki, Nathan J., and Mack, David L.
- Subjects
- *
HEART , *DYSTROPHIN , *CARDIAC contraction , *DUCHENNE muscular dystrophy , *HEART beat , *DRUG discovery , *MYOCARDIUM , *TISSUE engineering - Abstract
Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
22. Embryonic Stem Cells Are Redirected to Non-Tumorigenic Epithelial Cell Fate by Interaction with the Mammary Microenvironment.
- Author
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Boulanger, Corinne A., Bruno, Robert D., Mack, David L., Gonzales, Monica, Castro, Nadia P., Salomon, David S., and Smith, Gilbert H.
- Subjects
- *
CANCER research , *EPITHELIAL cells , *MAMMARY glands , *EMBRYONIC stem cell research , *CANCER prevention , *CARCINOGENESIS , *DEVELOPMENTAL biology , *CELL differentiation , *LABORATORY mice - Abstract
Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1∶5 and 1∶50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+) progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. We demonstrate that these progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
23. Witness to a Changing World.
- Author
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Mack, David L.
- Subjects
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MEMOIRS , *NONFICTION ,MODERN history, 1945-1989 - Abstract
The article reviews the book "Witness to a Changing World" by David D. Newsom.
- Published
- 2009
24. Absence of full-length dystrophin impairs normal maturation and contraction of cardiomyocytes derived from human-induced pluripotent stem cells.
- Author
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Pioner, J Manuel, Guan, Xuan, Klaiman, Jordan M, Racca, Alice W, Pabon, Lil, Muskheli, Veronica, Macadangdang, Jesse, Ferrantini, Cecilia, Hoopmann, Michael R, Moritz, Robert L, Kim, Deok-Ho, Tesi, Chiara, Poggesi, Corrado, Murry, Charles E, Childers, Martin K, Mack, David L, and Regnier, Michael
- Subjects
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PLURIPOTENT stem cells , *DYSTROPHIN , *INDUCED pluripotent stem cells , *DUCHENNE muscular dystrophy , *DYSTROPHIN genes - Abstract
Aims Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient. Methods and results The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG -CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80–100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils. Conclusions Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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25. Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease.
- Author
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Berry, Bonnie J., Smith, Alec S.T., Young, Jessica E., and Mack, David L.
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NEURONS , *GENE expression , *CELLS , *MOLECULAR genetics , *STEM cells - Abstract
One of the most profound advances in the last decade of biomedical research has been the development of human induced pluripotent stem cell (hiPSC) models for identification of disease mechanisms and drug discovery. Human iPSC technology has the capacity to revolutionize healthcare and the realization of personalized medicine, but differentiated tissues derived from stem cells come with major criticisms compared to native tissue, including variability in genetic backgrounds, a lack of functional maturity, and differences in epigenetic profiles. It is widely believed that increasing complexity will lead to improved clinical relevance, so methods are being developed that go from a single cell type to various levels of 2-D coculturing and 3-D organoids. As this inevitable trend continues, it will be essential to thoroughly understand the strengths and weaknesses of more complex models and to develop criteria for assessing biological relevance. We believe the payoff of robust, high-throughput, clinically meaningful human stem cell models could be the elimination of often inadequate animal models. To facilitate this transition, we will look at the challenges and strategies of complex model development through the lens of neurodegeneration to encapsulate where the disease-in-a-dish field currently is and where it needs to go to improve. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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- View/download PDF
26. ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy.
- Author
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Gartz, Melanie, Haberman, Margaret, Sutton, Jessica, Slick, Rebecca A., Luttrell, Shawn M., Mack, David L., and Lawlor, Michael W.
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NEMALINE myopathy , *MUSCLE weakness , *MITOCHONDRIA , *MUSCLE cells , *GENE expression , *MITOCHONDRIAL membranes , *MYOBLASTS - Abstract
Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study. • Directed differentiation of ACTA1 H40Y nemaline myopathy (NM) iPSCs into skeletal myotubes. • Nemaline myopathy iPSC-derived skeletal myocytes (NM-iSkM) do not display nemaline rods. • NM-iSkM show mitochondrial dysfunction under conditions of stress. • NM-iSkM are vulnerable to oxidative stress. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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27. Muscular dystrophy in a dish: engineered human skeletal muscle mimetics for disease modeling and drug discovery.
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Smith, Alec S.T., Davis, Jennifer, Lee, Gabsang, Mack, David L., and Kim, Deok-Ho
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DRUG development , *DRUG efficacy , *DRUG toxicity , *MUSCULAR dystrophy , *PLURIPOTENT stem cells , *PATHOLOGICAL physiology , *ANIMAL models in research - Abstract
Engineered in vitro models using human cells, particularly patient-derived induced pluripotent stem cells (iPSCs), offer a potential solution to issues associated with the use of animals for studying disease pathology and drug efficacy. Given the prevalence of muscle diseases in human populations, an engineered tissue model of human skeletal muscle could provide a biologically accurate platform to study basic muscle physiology, disease progression, and drug efficacy and/or toxicity. Such platforms could be used as phenotypic drug screens to identify compounds capable of alleviating or reversing congenital myopathies, such as Duchene muscular dystrophy (DMD). Here, we review current skeletal muscle modeling technologies with a specific focus on efforts to generate biomimetic systems for investigating the pathophysiology of dystrophic muscle. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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28. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.
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Padilla-Nash, Hesed M., McNeil, Nicole E., Yi, Ming, Nguyen, Quang-Tri, Hu, Yue, Wangsa, Danny, Mack, David L., Hummon, Amanda B., Case, Chanelle, Cardin, Eric, Stephens, Robert, Difilippantonio, Michael J., and Ried, Thomas
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ANEUPLOIDY , *ONCOGENES , *GENE amplification , *EPITHELIAL cells , *CELL transformation , *CANCER relapse , *STIMULUS & response (Biology) - Abstract
Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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29. Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering.
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Mirmalek-Sani, Sayed-Hadi, Orlando, Giuseppe, McQuilling, John P., Pareta, Rajesh, Mack, David L., Salvatori, Marcus, Farney, Alan C., Stratta, Robert J., Atala, Anthony, Opara, Emmanuel C., and Soker, Shay
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EXTRACELLULAR matrix , *PANCREAS , *ISLANDS of Langerhans , *BIOENGINEERING , *TRANSPLANTATION of organs, tissues, etc. , *TISSUE scaffolds , *TREATMENT of diabetes - Abstract
Abstract: Emergent technologies of regenerative medicine have the potential to overcome the limitations of organ transplantation by supplying tissues and organs bioengineered in the laboratory. Pancreas bioengineering requires a scaffold that approximates the biochemical, spatial and vascular relationships of the native extracellular matrix (ECM). We describe the generation of a whole organ, three-dimensional pancreas scaffold using acellular porcine pancreas. Imaging studies confirm that our protocol effectively removes cellular material while preserving ECM proteins and the native vascular tree. The scaffold was seeded with human stem cells and porcine pancreatic islets, demonstrating that the decellularized pancreas can support cellular adhesion and maintenance of cell functions. These findings advance the field of regenerative medicine towards the development of a fully functional, bioengineered pancreas capable of establishing and sustaining euglycemia and may be used for transplantation to cure diabetes mellitus. [Copyright &y& Elsevier]
- Published
- 2013
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30. Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland.
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Strizzi, Luigi, Mancino, Mario, Bianco, Caterina, Raafat, Ahmed, Gonzales, Monica, Booth, Brian W., Watanabe, Kazuhide, Nagaoka, Tadahiro, Mack, David L., Howard, Beatrice, Callahan, Robert, Smith, Gilbert H., and Salomon, David S.
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MAMMARY glands , *LABORATORY mice , *LACTATION , *WESTERN immunoblotting , *EPITHELIUM , *EPITHELIAL cells - Abstract
Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland. J. Cell. Physiol. 216: 824–834, 2008, © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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31. New cell lines from mouse epiblast share defining features with human embryonic stem cells.
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Tesar, Paul J., Chenoweth, Josh G., Brook, Frances A., Davies, Timothy J., Evans, Edward P., Mack, David L., Gardner, Richard L., and McKay, Ronald D. G.
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EMBRYONIC stem cells , *CELL populations , *CELL lines , *TRANSCRIPTION factors , *GENE expression , *GERM cells - Abstract
The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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32. Astrocyte-derived extracellular vesicles enhance the survival and electrophysiological function of human cortical neurons in vitro.
- Author
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Chun, Changho, Smith, Alec S.T., Kim, Hyejin, Kamenz, Dana S., Lee, Jung Hyun, Lee, Jong Bum, Mack, David L., Bothwell, Mark, Clelland, Claire D., and Kim, Deok-Ho
- Subjects
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NEURAL stem cells , *PLURIPOTENT stem cells , *EXTRACELLULAR vesicles , *HEAT shock proteins , *SURVIVAL analysis (Biometry) , *INDUCED pluripotent stem cells , *NEURONS - Abstract
Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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33. rAAV-related therapy fully rescues myonuclear and myofilament function in X-linked myotubular myopathy.
- Author
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Ross, Jacob A., Tasfaout, Hichem, Levy, Yotam, Morgan, Jennifer, Cowling, Belinda S., Laporte, Jocelyn, Zanoteli, Edmar, Romero, Norma B., Lowe, Dawn A., Jungbluth, Heinz, Lawlor, Michael W., Mack, David L., and Ochala, Julien
- Subjects
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CYTOPLASMIC filaments , *MUSCLE weakness , *MUSCLE diseases , *CONTRACTILE proteins , *SKELETAL muscle , *ANIMAL rescue - Abstract
X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 1013 vg kg−1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
34. Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates.
- Author
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Pioner, Josè Manuel, Santini, Lorenzo, Palandri, Chiara, Martella, Daniele, Lupi, Flavia, Langione, Marianna, Querceto, Silvia, Grandinetti, Bruno, Balducci, Valentina, Benzoni, Patrizia, Landi, Sara, Barbuti, Andrea, Ferrarese Lupi, Federico, Boarino, Luca, Sartiani, Laura, Tesi, Chiara, Mack, David L., Regnier, Michael, Cerbai, Elisabetta, and Parmeggiani, Camilla
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PLURIPOTENT stem cells , *INDUCED pluripotent stem cells , *CALCIUM , *SARCOPLASMIC reticulum , *OPTICAL measurements - Abstract
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to β-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
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