Ventricle Of Neonatal Mice Research Articles

Dynamic upregulation of retinoic acid signal in the early postnatal murine heart promotes cardiomyocyte cell cycle exit and maturation

The adult mammalian heart has extremely limited cardiac regenerative capacity. Most cardiomyocytes live in a state of permanent cell-cycle arrest and are unable to re-enter the cycle. Cardiomyocytes switch from cell proliferation to a maturation state during neonatal development. Although several signaling pathways are involved in this transition, the molecular mechanisms by which these inputs coordinately regulate cardiomyocyte maturation are not fully understood. Retinoic acid (RA) plays a pivotal role in development, morphogenesis, and regeneration. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. We found that mRNA expression of aldehyde dehydrogenase 1 family member A2 (Aldh1a2), which encodes the key enzyme for synthesizing all-trans retinoic acid (ATRA) and is an important regulator for RA signaling, was transiently upregulated in neonatal mouse ventricles. Single-cell transcriptome analysis and immunohistochemistry revealed that Aldh1a2 expression was enriched in cardiac fibroblasts during the early postnatal period. Administration of ATRA inhibited cardiomyocyte proliferation in cultured neonatal rat cardiomyocytes and human cardiomyocytes. RNA-seq analysis indicated that cell proliferation-related genes were downregulated in prenatal rat ventricular cardiomyocytes treated with ATRA, while cardiomyocyte maturation-related genes were upregulated. These findings suggest that RA signaling derived from cardiac fibroblasts is one of the key regulators of cardiomyocyte proliferation and maturation during neonatal heart development.

Pyk2 inhibition attenuates hypoxic-ischemic brain injury in neonatal mice.

Newborns suffering from hypoxia-ischemia (HI) brain injury still lack effective treatment. Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase, which is highly correlated with transient ischemic brain injury in adult. In this study, we investigated the role of Pyk2 in neonatal HI brain injury. HI was induced in postnatal day 7 mouse pups by unilateral common carotid artery ligation followed by hypoxic exposure. Pyk2 interference lentivirus (LV-Pyk2 shRNA) was constructed and injected into unilateral cerebral ventricle of neonatal mice before HI. Infarct volume, pathological changes, and neurological behaviors were assessed on postnatal day 8-14. We showed that the phosphorylation level of Pyk2 was significantly increased in neonatal brain after HI, whereas LV-Pyk2 shRNA injection significantly attenuated acute HI brain damage and improved neurobehavioral outcomes. In oxygen-glucose deprivation-treated cultured cortical neurons, Pyk2 inhibition significantly alleviated NMDA receptor-mediated excitotoxicity; similar results were also observed in neonatal HI brain injury. We demonstrated that Pyk2 inhibition contributes to the long-term cerebrovascular recovery assessed by laser speckle contrast imaging, but cognitive function was not obviously improved as evaluated in Morris water maze and novel object recognition tests. Thus, we constructed lentiviral LV-HIF-Pyk2 shRNA, through which HIF-1α promoter-mediated interference of Pyk2 would occur during the anoxic environment. Intracerebroventricular injection of LV-HIF-Pyk2 shRNA significantly improved long-term recovery of cognitive function in HI-treated neonatal mice. In conclusion, this study demonstrates that Pyk2 interference protects neonatal brain from hypoxic-ischemic injury. HIF-1α promoter-mediated hypoxia conditional control is a useful tool to distinguish between hypoxic period and normal period. Pyk2 is a promising drug target for potential treatment of neonatal HI brain injury.

Overexpression of HGF/MET axis along with p53 inhibition induces de novo glioma formation in mice.

BackgroundAberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive.MethodsUsing a well-established Sleeping Beauty (SB) transposon strategy, we injected human HGF and MET cDNA together with a short hairpin siRNA against Trp53 (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs.ResultsMixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres’ proliferation and MET/MAPK/AKT signaling.ConclusionsOverexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

Postnatal nectin-3 knockdown induces structural abnormalities of hippocampal principal neurons and memory deficits in adult mice.

The early postnatal stage is a critical period of hippocampal neurodevelopment and also a period of high vulnerability to adverse life experiences. Recent evidence suggests that nectin‐3, a cell adhesion molecule, mediates memory dysfunction and dendritic alterations in the adult hippocampus induced by postnatal stress. But it is unknown whether postnatal nectin‐3 reduction alone is sufficient to alter hippocampal structure and function in adulthood. Here, we down regulated hippocampal expression of nectin‐3 and its heterophilic adhesion partner nectin‐1, respectively, from early postnatal stage by injecting adeno‐associated virus (AAV) into the cerebral lateral ventricles of neonatal mice (postnatal day 2). We found that suppression of nectin‐3, but not nectin‐1, expression from the early postnatal stage impaired hippocampus‐dependent novel object recognition and spatial object recognition in adult mice. Moreover, AAV‐mediated nectin‐3 knockdown significantly reduced dendritic complexity and spine density of pyramidal neurons throughout the hippocampus, whereas nectin‐1 knockdown only induced the loss of stubby spines in CA3. Our data provide direct evidence that nectins, especially nectin‐3, are necessary for postnatal hippocampal development of memory functions and structural integrity.

Survival and Proliferation of Neural Progenitor–Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism

Purpose: One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPC). The development and molecular signature of NPC-derived glioblastomas were analyzed and the therapeutic effect of blocking CXCL12 was tested.Experimental Design: Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis, and expression of proteins regulating survival and cell-cycle progression.Results: Tumors induced from NPCs display histologic features of human glioblastoma and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell-cycle progression, downregulates molecules regulating survival and proliferation, and also blocks the hypoxic induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation.Conclusions: This study demonstrates that the CXCL12/CXCR4 axis operates in glioblastoma cells under hypoxic stress via an autocrine-positive feedback mechanism, which promotes survival and cell-cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced glioblastoma progression, prevent resistance to treatment, and recurrence of the disease. Clin Cancer Res; 23(5); 1250-62. ©2016 AACR.

Abstract 3009: ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM

Abstract Pediatric Glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most children with this diagnosis will not survive longer than two years. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in neonatal mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors displayed evidence of telomeric lengthening using telomeric FISH assay for alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage, by gH2A.X. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced tumor growth by luminescence, with some long-term survivors. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased genetic instability and response to DNA-damaging therapy. Based on these results, we have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival. Supported by St. Baldrick's Fellowship and Alex's Lemonade Stand /Northwest Mutual Young Investigator Award to CK and NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Daniel Thomas, Felipe J. Nunez, Marta Dzaman, Johnny Krasinkiewicz, Rosie Lemons, Neha Kamran, Flor Mendez, Soyeon Roh, David Ferguson, Pedro R. Lowenstein, Maria G. Castro. ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2015-3009

A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles

The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage—neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines.

HG-02 * ATRX LOSS PROMOTES TUMOR GROWTH AND IMPAIRS GENETIC STABILITY IN GLIOBLASTOMA

Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced by injecting plasmids encoding SB transposase/luciferase, shp53 and NRAS, with or without shATRX, into the lateral ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human GBM. The loss of ATRX was localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX significantly reduced median survival (p = 0.0032). Loss of ATRX in mouse GBM impaired genetic stability using multiple assays: (1) ATRX-deficient tumors were more likely to develop microsatellite instability, a hallmark of impaired DNA-damage repair; (2) ATRX-deficient tumors displayed evidence of telomeric lengthening, and (3) ATRX loss impaired non-homologous end-joining (NHEJ) utilizing a GFP reporter plasmid system. Analysis of human pediatric (St. Jude's/Washington University) and adult (UCSF) glioma datasets confirmed increased number of somatic nucleotide mutations in tumors with ATRX mutation. Finally, treatment of primary mouse GBM cell cultures showed that ATRX-deficient tumor cells were significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced early tumor growth by luminescence. This data demonstrates the role of ATRX in GBM tumor progression, treatment response and loss of tumor genetic stability, and provides a platform for future development of targeted therapy for pediatric and young adult patients with ATRX-deficient GBM.

PM-07 * LOSS OF ATRX DECREASES SURVIVAL AND IMPROVES RESPONSE TO DNA DAMAGING AGENTS IN A NOVEL MOUSE MODEL OF GLIOBLASTOMA

Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat. ATRX is a histone chaperone protein that is mutated primarily in younger patients with GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to DNA damaging agents. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation had trend towards improved survival (p= 0.06), with some long-term survivors. Treated ATRX-deficient tumor cells showed greater evidence of double-stranded DNA breakage, by gH2A.X. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased mutation frequency and response to DNA-damaging therapy. We have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival.

PM-21 * A REVERSE GENETIC SCREEN FOR DRIVERS OF GBM

INTRODUCTION: Glioblastoma multiforme (GBM) is the most malignant and most common primary brain tumor. There is no curative chemotherapeutic regimen for GBM, and its invasiveness greatly limits efficacy of surgery and radiation therapy. Molecular drivers of GBM growth, maintenance, and invasion need to be identified to develop effective therapies. METHODS: We selected 50 genes found to be overexpressed in a high proportion of GBM in comprehensive sequencing and gene expression analysis studies by The Cancer Genome Atlas (TCGA) Research Network and the Howard Hughes Medical Institute (Parsons et al 2008). We hypothesized a common subset of these genes directly promote tumor initiation or maintenance downstream of initiating genetic alterations, and screened them in vivo. A cDNA for each of these genes and SV40 large T antigen (TAg) was cloned into a separate Sleeping Beauty (SB) transposon plasmid. The cDNA delivery plasmids were stereotactically injected into the lateral ventricles of neonatal mice in batches of 9, along with the TAg vector to facilitate tumor formation and a SB transposase expression plasmid containing a firefly luciferase transposon. We monitored tumor development by luciferase imaging. RESULTS: Tumors developed in a subset of mice, the majority of which were highly infiltrative with vascular proliferation and histologically resembled grade 3 glioma. Injection of the HIF1a cDNA was associated with infiltrative tumors in our model and has been implicated in glioma infiltration previously. UBE2C, CCNE2, and EZH2 cDNAs were associated with reduced tumor latency and may drive glioma cell proliferation. We are testing these cDNAs individually in vivo and in vitro to confirm their roles in driving infiltration and proliferation. CONCLUSIONS: We used a SB transposon-based reverse genetic mouse model to screen for common GBM drivers. Of the 50 genes screened, we identified 4 strong candidates which may drive tumor cell proliferation and infiltration.

PM-14 * MODELING GBM IN MICE USING THE CRISPR NUCLEASE SYSTEM TO FUNCTIONALLY VALIDATE NOVEL CANDIDATE DRIVER GENES AND TEST NEW THERAPIES

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor and has a dismal survival rate of less than 20% in pediatric patients and less than 5% in adults. Identifying and functionally validating driver genes is needed to develop novel therapies to better treat GBM. To this end, we developed a novel method to induce de novo GBM in mice via in vivo gene transfer of the CRISPR nuclease system to functionally validate and/or screen candidate drivers. The CRISPR system is composed of the Cas9 nuclease that uses a RNA guide molecule (gRNA) to identify specific DNA sequences and induce double strand breaks (DSBs). These DSB are repaired by either the error prone process of non-homologous end joining (NHEJ) or perfectly repaired by homologous recombination (HR), allowing the opportunity for gene knockout (KO) or introduction of new sequences into the genome, respectively. To test our system, we utilized information from a pre-validated set of genes shown to induce GBM via in vivo gene transfer of Sleeping Beauty transposons harboring NRASG12V and shTrp53. Thus, Polyethylenimine (PEI) complexed Cas9, Trp53-gRNA, SB100X transposase, and NRASG12V transposon plasmids were injected into the lateral ventricles of neonatal mice and aged for tumor development. Animals injected with Cas9 and Trp53-gRNA or SB100X and NRASG12V alone did not develop tumors whereas all animals receiving both developed rapid onset GBM in 60 ± 16 days. Analysis of the Trp53-gRNA target site from tumors demonstrated clonal frame-shift mutations. These results demonstrate that the CRISPR is highly efficient in vivo to KO gene function. Currently, we are replacing the NRASG12V transposon with the use of HR to incorporate the G12V mutation at the endogenous Nras locus. Future studies will focus on validating candidate GBM driver genes identified from human genomics data and forward genetic screens and attempts to model GBM subgroups.

Abstract 995: Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma

Abstract Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most patients will die within the first two years of receiving this diagnosis. ATRX is a histone chaperone protein that is mutated primarily in adolescent GBMs. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. In this study, we determined the contribution of ATRX knockdown to GBM formation and treatment response in a novel mouse model of GBM. Using the second-generation shRNA-mir library, we cloned an ATRX knockdown sequence into a plasmid with flanking sequences recognized by the Sleeping Beauty (SB) transposase for insertion into host genomic DNA. Glioblastomas were induced in mice using the SB transposase system injecting plasmids encoding luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Uptake of plasmid DNA as well as development of intracranial tumors was monitored by bioluminescence. When animals showed symptoms of tumor burden they were euthanized and brains were processed for histological evaluation or placed in culture with neural stem cell media (with EGF and FGF supplementation). Tumors in both groups (with or without shATRX) showed histological hallmarks of human grade IV glioblastoma. The loss of ATRX was confirmed by IHC, and was specifically localized within tumors generated with the shATRX plasmid and not in the tumors generated with shp53 and NRAS alone, nor in the adjacent normal cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p=0.012). Tissue was analyzed by FISH telomere probe as ATRX loss in human tumors is associated with alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to show chromosomal aneuploidy (p=0.015) by telomere FISH. Cell lines generated from ATRX-deficient tumors were confirmed to have reduction of ATRX expression. Tumor cell lines (with and without ATRX loss) were plated, treated at 24 hours with intervention or control, and analyzed for viability at 72 hours. ATRX-deficient tumor cells were significantly (p≤0.005) more sensitive to DNA damaging agents, including: (1) 5-FU, (2) doxorubicin, (3) UV irradiation, and (4) adenoviral thymidine kinase with ganciclovir; with the notable exception of temozolomide (p=0.86), which is the standard of care for treatment of pediatric GBM. Loss of ATRX in a mouse model hastens glioblastoma formation and decreases survival. In addition, loss of ATRX leads to aneuploidy and improved response to DNA damaging agents, providing possible targeted therapies for tumors with this mutation. This mouse model prospectively validates ATRX as a tumor suppressor in pediatric GBM for the first time in an animal model; and provides a platform for analysis of relevant pathways and development of potential novel therapies. Supported by NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Marta Dzaman, Rosie Lemons, Daniel Thomas, Maria G. Castro, Pedro R. Lowenstein. Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 995. doi:10.1158/1538-7445.AM2014-995

Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

SummaryStem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications.

Capsid Serotype and Timing of Injection Determines AAV Transduction in the Neonatal Mice Brain

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Transplantation of CD15-enriched murine neural stem cells increases total engraftment and shifts differentiation toward the oligodendrocyte lineage.

Neural stem cell (NSC) transplantation is a promising therapeutic approach for neurological diseases. However, only a limited number of cells can be transplanted into the brain, resulting in relatively low levels of engraftment. This study investigated the potential of using a cell surface marker to enrich a primary NSC population to increase stable engraftment in the recipient brain. NSCs were enriched from the neonatal mouse forebrain using anti-CD15 (Lewis X antigen, or SSEA-1) in a "gentle" fluorescence-activated cell sorting protocol, which yielded >98% CD15-positive cells. The CD15-positive cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, after withdrawal of growth factors, demonstrating multipotentiality. CD15-positive cells were expanded in vitro and injected bilaterally into the ventricles of neonatal mice. Cells from enriched and unenriched donor populations were found throughout the neuraxis, in both neurogenic and non-neurogenic regions. Total engraftment was similar at 7 days postinjection, but by 28 days postinjection, after brain organogenesis was complete, the survival of donor cells was significantly increased in CD15-enriched grafts over the unenriched cell grafts. The engrafted cells were heterogeneous in morphology and differentiated into all three neural lineages. Furthermore, in the CD15-enriched grafts, there was a significant shift toward differentiation into oligodendrocytes. This strategy may allow better delivery of therapeutic cells to the developing central nervous system and may be particularly useful for treating diseases involving white matter lesions.

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

T-type Ca 2+ channel blockers prevent cardiac cell hypertrophy through an inhibition of calcineurin–NFAT3 activation as well as L-type Ca 2+ channel blockers

T-type Ca 2+ channels (TCCs) are involved in cardiac cell growth and proliferation in cultured cardiomyocytes. Underlying molecular mechanisms are not well understood. In this study, we investigated the role of TCCs in signal transduction in cardiac hypertrophy compared with L-type Ca 2+ channels (LCCs). Cardiomyocytes dissociated from neonatal mouse ventricles were cultured until stabilization. Cell hypertrophy was induced by reapplication of 1% fatal bovine serum (FBS) following a period (24 h) of FBS depletion. Cell surface area increased from 862 ± 73 μm 2 to 2153 ± 131 μm 2 by FBS stimulation in control (250 ± 1.8%). T-type Ca 2+ current ( I CaT) was inhibited dose-dependently by kurtoxin (KT) and efonidipine (ED) with IC 50 0.07 μM and 3.2 μM, respectively in whole-cell voltage clamp. On the other hand, 1 μM KT which inhibits I CaT over 90% did not effect on L-type Ca 2+ current ( I CaL). 10 μM ED had the ability of I CaL blockade as well as that of I CaT blockade. 3 μM nisoldipine (ND) suppressed I CaL by over 80%. The increase in cell surface area following reapplication of FBS as observed in control (250 ± 1.8%) was significantly reduced in the presence of 1 µM KT (216 ± 1.2%) and virtually abolished in the presence of 10 µM ED (97 ± 0.8%) and 3 μM ND (80 ± 1.1%). Hypertrophy was associated with an increase in BNP mRNA of 316 ± 3.6% in control and this increase was reduced as well in the presence of 1 µM KT (254 ± 1.8%) and almost abolished in the presence of 10 µM ED (116 ± 1.1%) and 3 μM ND (93 ± 0.8%). Immunolabeling showed that translocation of nuclear factor of activated T cells (NFAT3) into the nucleus in response to FBS stimulation was markedly inhibited by either KT or ED as well as ND. Calcineurin phosphatase activity was upregulated 2.2-fold by FBS, but KT, ED and ND decreased this upregulation (1.7-fold, 0.8-fold, and 0.7-fold with KT, ED and ND respectively). These results suggest that blockade of Ca 2+ entry into cardiomyocytes via TCCs may block pathophysiological signaling pathways leading to hypertrophy as well as via LCCs. The mechanism may be the inhibition of calcineurin-mediated NFAT3 activation resulting in prevention of its translocation into the nucleus.

Complete Correction of Enzymatic Deficiency and Neurochemistry in the GM1-gangliosidosis Mouse Brain by Neonatal Adeno-associated Virus–mediated Gene Delivery

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal), and characterized by accumulation of GM1-ganglioside and GA1 in the brain. Here we examined the effect of neonatal intracerebroventricular (i.c.v.) injection of an adeno-associated virus (AAV) vector encoding mouse betagal on enzyme activity and brain GSL content in GM1-gangliosidosis (betagal(-/-)) mice. Histological analysis of betagal distribution in 3-month-old AAV-treated betagal(-/-) mice showed that enzyme was present at high levels throughout the brain. Biochemical quantification showed that betagal activity in AAV-treated brains was 7- to 65-fold higher than in wild-type controls and that brain GSL levels were normalized. Cerebrosides and sulfatides, which were reduced in untreated betagal(-/-) mice, were restored to normal levels by AAV treatment. In untreated betagal(-/-) brains, cholesterol was present at normal levels but showed abnormal cellular distribution consistent with endosomal/lysosomal localization. This feature was also corrected in AAV-treated mice. The biochemical and histological parameters analyzed in this study showed that normal brain neurochemistry was achieved in AAV-treated betagal(-/-) mice. Therefore we show for the first time that neonatal AAV-mediated gene delivery of lysosomal betagal to the brain may be an effective approach for treatment of GM1-gangliosidosis.

Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice.

The precise localization and role of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP(3)Rs underlie modifications of cellular responses that lead to changes in gene expression in other cells. However, how changes in cytosolic calcium alter gene expression in cardiac cells is unknown. Moreover, it is unclear how changes in cytosolic calcium that alter gene expression do so independently of effects of calcium on other cellular functions, such as contraction. Here we show that InsP(3)R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP(3)Rs are associated with the nucleus and that activation of type 2 InsP(3)Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal alpha-actin. Type 2 InsP(3)Rs are also in striations. Activation of InsP(3)Rs with adenophostin A in permeabilized cells induced calcium release in the nuclear domain and other regions of the cell away from the nucleus. Agonist-induced increase in gene expression and calcium release were blocked by the InsP(3)R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP(3)Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP(3)Rs in the nuclear domain provides a direct mechanism for the control of gene expression, whereas release of calcium in the cytoplasm may modulate other processes, such as contraction.

High-efficiency, long-term cardiac expression of foreign genes in living mouse embryos and neonates.

The development of improved strategies for efficient and reproducible in vivo gene transfer into the murine heart will ultimately allow the intersection of somatic and germline gene transfer strategies to study complex features of cardiac biology and diseases. For embryonic gene transfer, an adenovirus vector expressing beta-galactosidase was injected in utero into the ventricular cavity of living embryos via microsurgical approaches. The injected embryos were developed to term, and efficient expression of the transgene was detected in all cell types in the heart. For postnatal cardiac gene transfer, adenovirus was injected into the cardiac ventricle of neonatal mice, resulting in efficient expression of the transgene in the outer layer of the myocardium as well as cardiomyocytes in the middle and inner layers of the cardiac wall. Mice examined after 3 weeks displayed a pattern of expression that completely mimicked the pattern seen after 3 days, and gene expression was also found after 6 months. The infected myocytes can be identified by coinfection of an adenovirus expressing green fluorescent protein without affecting their normal physiological function. We have developed a new strategy to achieve efficient and long-term foreign gene expression in both embryonic and postnatal mouse myocardium via direct intracardiac injection of recombinant adenovirus. The strategy should allow the functional assessment of the expression of dominantly acting exogenous genes, overexpression of wild-type genes, and Cre recombinase-mediated gene ablations at the single-cell level in the context of the intact adult mouse myocardium.