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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.

Summary Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention., Highlights • DMD hiPSC-CMs exhibit aberrant calcium handling and defective force generation • DMD hiPSC-CMs undergo proliferation-independent telomere shortening • Telomere shortening activates the p53 DNA damage pathway • Telomere shortening in DMD hiPSC-CMs is contraction dependent, In this article, Chang, Blau, and colleagues show that Duchenne muscular dystrophy (DMD) iPSC-derived cardiomyocytes exhibit proliferation-independent telomere shortening, p53 activation and mitochondrial dysfunction.

Gene editing technologies hold great potential to enhance our ability to model inheritable neurodegenerative diseases. Specifically, engineering multiple amyotrophic lateral sclerosis (ALS) mutations into isogenic cell populations facilitates determination of whether different causal mutations cause pathology via shared mechanisms, and provides the capacity to separate these mechanisms from genotype-specific effects. As gene-edited, cell-based models of human disease become more commonplace, there is an urgent need to verify that these models constitute consistent and accurate representations of native biology. Here, commercially sourced, induced pluripotent stem cell-derived motor neurons from Cellular Dynamics International, edited to express the ALS-relevant mutations TDP-43M337V and TDP-43Q331K were compared with in-house derived lines engineered to express the TDP-43Q331K mutation within the WTC11 background. Our results highlight electrophysiological and mitochondrial deficits in these edited cells that correlate with patient-derived cells, suggesting a consistent cellular phenotype arising from TDP-43 mutation. However, significant differences in the transcriptomic profiles and splicing behavior of the edited cells underscores the need for careful comparison of multiple lines when attempting to use these cells as a means to better understand the onset and progression of ALS in humans.

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.

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.

The global demand for fish is rising and projected to increase for years to come. However, there is uncertainty whether this increased demand can be met by the conventional approaches of capture fisheries and fish farming because of wild stock depletion, natural resource requirements, and environmental impact concerns. One proposed complementary solution is to manufacture the same meat directly from fish cells, as cell-based fish. More than 30 ventures are competing to commercialize cell-based meat broadly, but the field lacks a foundation of shared scientific knowledge, which threatens to delay progress. Here, we recommend taking a research-focused, more open and collaborative approach to cell-based fish meat development that targets lean fish and an unlikely but very attractive candidate for accelerating research and development, the zebrafish. Although substantial work lies ahead, cell-based meat technology could prove to be a more efficient, less resource-intensive method of producing lean fish meat.

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Previous work from our laboratory on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrects structural abnormalities within the muscle and restores contractile function, with affected dogs surviving more than four years post injection. This exceptional therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology, and to what extent the whole muscle transcriptome is restored to normal after gene transfer. Herein, RNA-sequencing was used to examine the transcriptomes of the Biceps femoris and Vastus lateralis in a previously-described canine cohort showing dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice, but also identified new transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created new metrics to pinpoint potential biomarkers of disease progression and correction.

Given the limited availability of tissue, especially brain tissue, for neurological diseases and disorders research, the development of alternative biological tools for investigations of underlying molecular and genetic mechanisms is imperative. One important resource for this task is the large repositories that bank immortalized blood cells (i.e. lymphoblastoid cell lines; LCLs) from affected individuals and their unaffected family members. These repositories document demographic, phenotypic, and, in some cases, genotypic information about the donors and thus provide a ready-made sample source for hypothesis testing. Importantly, patient-specific LCLs can be used to generate induced pluripotent stem cells (iPSC) that, in turn, can be used to create specific cell types for use in mechanistic studies. To investigate this concept further, LCLs from two males (proband and sibling) were obtained from one such repository, the Autism Genetics Resource Exchange (AGRE), and iPSCs were generated by transfection with Epi5 Episomal iPSC reprogramming plasmids. Characterization of the resultant cell lines by PCR, RT-PCR, immunocytochemistry, karyotyping, and the Taqman® human pluripotent stem cell Scorecard™ Panel, was used to provide evidence of endogenous pluripotency and then to evaluate the trilineage potential of four representative clones. Results indicated that all four iPSC lines were initially pluripotent and displayed the trilineage potential predictive for successful differentiation to mesoderm, endoderm, or ectoderm-derived cell types. Compared to other published protocols, this study details a somewhat simplified approach, used here specifically for the generation and characterization of induced pluripotent stem cells from well-characterized and banked LCLs., Lymphoblastoid cell lines, Induced pluripotent stem cells, Reprogramming, Epstein-Barr virus.

Background: Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. Methods and Results: Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide–cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore, nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. Conclusion: Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy.

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer unprecedented opportunities to study inherited heart conditions in vitro, but are phenotypically immature, limiting their ability to effectively model adult-onset diseases. Cardiomyopathy is becoming the leading cause of death in patients with Duchenne muscular dystrophy (DMD), but the pathogenesis of this disease phenotype is not fully understood. Therefore, we aimed to test whether biomimetic nanotopography could further stratify the disease phenotype of DMD hiPSC-CMs to create more translationally relevant cardiomyocytes for disease modeling applications. We found that anisotropic nanotopography was necessary to distinguish structural differences between normal and DMD hiPSC-CMs, as these differences were masked on conventional flat substrates. DMD hiPSC-CMs exhibited a diminished structural and functional response to the underlying nanotopography compared to normal cardiomyocytes at both the macroscopic and subcellular levels. This blunted response may be due to a lower level of actin cytoskeleton turnover as measured by fluorescence recovery after photobleaching. Taken together these data suggest that DMD hiPSC-CMs are less adaptable to changes in their extracellular environment, and highlight the utility of nanotopographic substrates for effectively stratifying normal and structural cardiac disease phenotypes in vitro.

Introduction X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years. Methods We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. Results Four years following AAV-mediated gene therapy, gait, respiratory performance, neurological function and pathology in AAV-infused XLMTM dogs remained comparable to their healthy littermate controls despite a decline in VCN and muscle strength. Conclusions AAV-mediated gene transfer of MTM1 in young XLMTM dogs results in long-term expression of myotubularin transgene with normal muscular performance and neurological function in the absence of muscle pathology. These findings support a clinical trial in patients. Muscle Nerve 56: 943–953, 2017

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

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.

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.

Summary Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations., Highlights • The contractile properties of hiPSC-CM myofibrils have not been previously studied • hiPSC-CMs cultured on nanopatterned surfaces develop elongated, aligned myofibrils • hiPSC-CMs myofibrils have contractile properties similar to human fetal myofibrils • hiPSC-CMs can be used to study development of genetically based cardiac diseases, In this article, Pioner and colleagues reported contractile properties of isolated myofibrils from hiPSC-CMs with highly mature morphology. This approach permits quantitative assessment of maturation and contractile properties of hiPSC-CMs and can be used to study the development of contractile dysfunction in genetically based cardiac diseases. The authors present a patient-derived cell line carrying a novel familial cardiomyopathy MYH7 mutation (E848G).

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in a tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and to determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, binucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors on injection into immunocompromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process.

Objective The aim of this study was to determine if mesenchymal stem cells (MSCs) could be obtained from mandible marrow aspirates. Study design In 5 patients, 10 mL marrow aspirates were obtained from both the mandible and the iliac crest. Second passage MSCs were characterized by fluorescence-activated cell sorting (FACS) analysis using MSC-specific cell surface marker, and fifth-passage cells were differentiated into 3 mesenchymal tissues in vitro. Results Average total cell yields of MSCs were 2.8 ± 1.8 × 10 5 per 10 mL marrow aspirates from the mandible. Immunophenotypes of MSCs isolated from the mandible and iliac crest were highly similar, as indicated by FACS analysis. Differentiation into mesodermal cell types, such as osteocytes, chondrocytes, and adipocytes, was successfully achieved in all MSC isolates from all aspirates. Conclusions The MSCs can be isolated from the mandibular aspirates, providing an alternative accessible source of MSCs for the treatment of future dental and craniomaxillofacial diseases.

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.

International audience; Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) show great promise as autologousdonor cells to treat heart disease. A major technical obstacle to this approach is that availableinduction methods often produce heterogeneous cell population with low percentage of cardiomyocytes.Here we describe a cardiac enrichment approach using nonintegrating adeno-associated virus (AAV). Wefirst examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes.Results showed that AAV1 demonstrated the highest in vitro transduction efficiency among sevenwidely used serotypes. Next, differentiated iPSC derivatives were transduced with drug-selectable AAV1expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to57% in 2 weeks. Compared with other enrichment strategies such as integrative genetic selection, mitochondrialabeling, or surface marker cell sorting, this simple AAV method described herein bypassesantibody or dye labeling. These findings provide proof of concept for large-scale cardiomyocyte enrichmentby exploiting AAV’s intrinsic tissue tropism

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.

Homeobox genes are involved in neoplastic transformation of both epithelial and hemopoietic tissues. The divergent homeobox gene HEX is expressed in the anterior visceral endoderm during early mouse development and in some adult tissues of endodermal origin, including liver and thyroid. Whereas a role in leukemyogenesis has been proposed already, few data are available on the involvement of HEX in human epithelial tumors. Herein, we analyzed HEX expression and subcellular localization in a series of 55 human thyroid tumors and in several tumoral cell lines. HEX mRNA was detected by RT-PCR either in normal tissues or in thyroid adenomas and differentiated (papillary and follicular) carcinomas. HEX mRNA was also expressed in most undifferentiated carcinomas. Subcellular localization of HEX protein was investigated by immunohistochemistry. In normal tissues and adenomas, HEX protein was present both in nucleus and cytoplasm. In contrast, both differentiated and undifferentiated carcinomas, as well as the tumoral cell lines investigated, showed HEX protein only in the cytoplasm. These findings suggest that regulation of HEX entry in the nucleus of thyrocytes may represent a critical step during human thyroid tumorigenesis. (J Clin Endocrinol Metab 87: 1376 –1383, 2002)

International audience

International audience; Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

PubMed ID: 10597310, 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 if 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., Leukemia Research Foundation RO1 CA80728-01, MT-13608, The mAb 5E10 antibody was a kind gift of L de Jong. We are grateful for technical assistance and advice from Graeme W Carlile and Melanie J Dobson and for critical reading of the manuscript by Ari Melnick. KLB Borden acknowledges support from MRC MT-13608, RO1 CA80728-01 and Leukemia Research Foundation.

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.

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.

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.

The physical cues presented to stem cells by the substrate on or in which they exist have been shown to play a crucial role in regulation of their behavior. Until recently, most research has focused on the effects of substrate elasticity on differentiation capability rather than maintenance of long-term proliferation and plasticity. The main goal of the present study is to study the interaction of amniotic fluid-derived stem (AFS) cells with growth substrata of different elasticity, which may extend their application potentials. Here, we investigate the effects of elastic modulus (E′), on AFS cell proliferation, morphology, cell surface marker expression, and autocrine stimulation of cell migration. AFS cells cultured on substrates of different E′ exhibited significant changes in proliferation and morphology. Immunohistochemistry revealed increased expression of cell surface markers associated with mesenchymal stem cells (MSCs) (CD44, CD90, CD105, and N-cadherin) in cells cultured on softer substrates. Additionally, AFS cells cultured on softer substrates induced autocrine stimulation of migration. Therefore, tailoring the elastic modulus of biomaterials to specific stiffness values is an effective method to control stem cell properties, which may modulate the effectiveness of their therapeutic applications.

AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein.

The mammary gland is unique in that most of its development occurs after birth with dramatic changes in proliferation and differentiation taking place during puberty and pregnancy. Different subsets of mammary-specific stem/progenitor cells have been shown to drive the individual stages of mammary gland development, and their regulation requires coordination between localized signals and systemic hormones. That sophisticated integration and control is achieved through the function of the stem cell niche. The goal of this chapter is to review why somatic stem cells are thought to exist in the mouse mammary gland, how they are being isolated and assayed, how their fate is influenced by the surrounding microenvironment, and how aberrant regulation of this process might contribute to breast cancer. If the components of the niche could be defined, each might then be targeted as a method to modify the fate of stem or progenitor cells during normal organ regeneration or repaired after tumorigenesis has been initiated.

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 mammary 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 + cells expressed mammary-specific functions, including milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSCs and their progeny enter mammary epithelium–specific niches and adopt the function of similarly endowed mammary 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.

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.

Introduction Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein. Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas. Methods To validate that Int6sh has transforming activity in vivo, a transgenic mouse model was designed using the whey acidic protein (Wap) promoter to target expression of truncated Int6 to differentiating alveolar epithelial cells in the mammary gland. Microarray analyses were performed on normal, premalignant and malignant WapInt6sh expressing tissues. Results Mammary tumors developed in 42% of WapInt6sh heterozygous parous females at an average age of 18 months. In WapInt6sh mice, the contralateral mammary glands from both tumorous and non-tumorous tissues contained widespread focal alveolar hyperplasia. Only 4% of WapInt6sh non-breeding females developed tumors by 2 years of age. The Wap promoter is active only during estrus in the mammary tissue of cycling non-pregnant mice. Microarray analyses of mammary tissues demonstrated that Int6sh expression in the alveolar tissue altered the mammary transcriptome in a specific manner that was detectable even in the first pregnancy. This Int6sh-specific transcriptome pattern subsequently persisted in both the Int6sh-expressing alveolar hyperplasia and mammary tumors. These observations are consistent with the conclusion that WapInt6sh-expressing alveolar cells survive involution following the cessation of lactation, and subsequently give rise to the mammary tumors that arise in aging multiparous females. Conclusion These observations provide direct in vivo evidence that mammary-specific expression of the Int6sh truncation leads to persistence of alveolar hyperplasia with the accompanying increased predisposition to mammary tumorigenesis.

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.

Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown promise both in preclinical studies and clinical trials for a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD). Studies have shown that host immune responses against AAV can compromise vector delivery and prevent sustained therapeutic gene expression in animal models and humans. We developed and demonstrated a novel AAV production system that prevents aberrant packaging of the AAV capsid (cap) genes. Compared to conventional AAV production methods, our new approach eliminates unwanted in vivo expression of AAV capsid proteins and significantly reduces host immune responses. Here, we demonstrated in a pilot study that we were able to reduce a tripledrug immunosuppressive regimen to a mild 2-drug regimen while retaining sustained canine micro-dystrophin expression in CXMD dogs following large scale delivery of an AAV vector carrying the canine micro-dystrophin to a group of muscles in the hind limb.Amelioration of muscle pathology as a result of sustained expression of the therapeutic gene was demonstrated by histological analysis of muscle tissues and by non-invasive magnetic resonance imaging (MRI). Further, we determined functional benefit of micro-dystrophin expression by muscle force measurement and gait analysis. Significant improvement in muscle force, as indicated by more than 100% increase in strength (torque), was observed in the treated limb compared to that in the untreated limb. In conclusion, by combining newly improved AAV production and mild clinically applicable immunosuppression, the finding of long term robust dystrophin expression in CXMD dogs’ limb with significant functional improvement opens the possibility of translating these strategies to a human DMD trial, which can improve patients’ immediate quality of life.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

SummaryEnhancement of thrombolysis with combinations of tissue-type and single chain urokinase plasminogen activators (t-PA and scu-PA) has been demonstrated in vivo but has not been seen consistently in vitro. This study was designed to characterize interactions between t-PA and scu-PA with respect to rate of and extent of thrombolysis in vitro and to delineate mechanisms responsible. Combinations of t-PA and scu-PA at selected concentrations synergistically enhanced thrombolysis in vitro compared with thrombolysis induced by either activator alone. Enhanced thrombolysis did not occur at the expense of fibrin specificity since the extent of fibrinogenolysis and consumption of α2-antiplasmin were significantly less with synergistic combinations of t-PA and scu-PA compared with equi-effective concentrations of either activator alone. Attenuation of complex formation of t-PA and two chain u-PA (tcu-PA), formed from scu-PA, with plasma proteins did not appear to contribute to enhancement of thrombolysis as assessed by fibrin autography. Binding of 125I-t-PA to thrombi was increased by 27% at 1 hr and by 21% at 2 hr in the presence of scu-PA (p