Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT.

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (µCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.

Attenuated reovirus displays oncolysis with reduced host toxicity.

BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.

Loss of discordant cells during micro-mass differentiation of embryonic stem cells into the chondrocyte lineage.

Embryonic stem cells (ESCs) have attracted particular interest in regenerative medicine because of their unlimited self-renewal and multipotentiality for differentiation. Spontaneous differentiated ESCs display heterogeneous multipotent cell populations and generate teratomas in vivo, with process by which ESCs differentiate into specific lineages remaining unclear. In this study, we focused on the in vitro chondrocyte differentiation of ESCs through micro-mass without using an embryoid body (EB) step and observed the unique characteristics of cartilage formation coupled with endochondral ossification in vivo. This approach resulted in an aggressive loss of discordant cells by apoptosis, which was accompanied by significant changes in gene expression during the course of ESC differentiation into chondrocytes. Unlike EB formation where discordant cells remain trapped within aggregates, micro-mass permits cells to die, leave the group and/or form a new group in response to changes in gene expression. Our observations suggest that the cell death that accompanies ESC micro-mass differentiation helps purify a terminally differentiated cell population and selects for targeted end points within a suitable microenvironment.

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.

RED: the analysis, management and dissemination of expressed sequence tags.

The Rancourt EST Database (RED) is a web-based system for the analysis, management, and dissemination of expressed sequence tags (ESTs). RED represents a flexible template DNA sequence database that can be easily manipulated to suit the needs of other laboratories undertaking mid-size sequencing projects.

Bacteriophage gene targeting vectors generated by transplacement.

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.

Regulation of the strypsin-related proteinase ISP2 by progesterone in endometrial gland epithelium during implantation in mice.

Hormones prepare the uterus for the arrival and subsequent invasion of the embryo during pregnancy. Extracellular matrix-degrading proteinases and their inhibitors are involved in this integration process. Recent genetic evidence indicates that there is redundancy within the implantation proteinase cascade, indicating that additional proteinases may be involved. Recently, we described a novel implantation serine proteinase (ISP1) gene that encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The evidence presented in the present study indicates that a second proteinase secreted from the uterus also participates in lysis of the zona pellucida. A second implantation serine proteinase gene (ISP2) was isolated, which encodes a related secreted tryptase expressed specifically within uterine endometrial glands. In pseudopregnancy, ISP2 gene expression is dependent on progesterone priming and is inhibited by the antiprogestin RU486. On the basis of similarities between ISP2 gene expression and that of a progesterone-regulated luminal proteinase associated with lysis of the zona pellucida, it is possible that the strypsin-related protein, ISP2, may encode a zona lysin proteinase.

A novel murine tryptase involved in blastocyst hatching and outgrowth.

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.

Neurons from stem cells: implications for understanding nervous system development and repair.

Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Isolation and characterization of a calcium channel gene, Cacna1f, the murine orthologue of the gene for incomplete X-linked congenital stationary night blindness.

The mutant L-type calcium channel alpha(1)-subunit gene, CACNA1F, was recently identified as the gene responsible for incomplete X-linked congenital stationary night blindness. The 6070-bp mRNA transcript is predicted to encode a 1977-amino-acid pore-forming protein with cytoplasmic amino- and carboxyl-termini separated by four homologous repeat domains, each consisting of six transmembrane segments. CACNA1F has been shown to be preferentially expressed in the retina, indicative of a specific functional role in visual processing. We have established the complete sequence of the murine orthologue of CACNA1F, namely Cacna1f. The total length of the mRNA transcript of the murine gene was established to be 6080 bp with an open reading frame that translates into a 1985-amino-acid protein. Cacna1f is highly homologous to the human sequence, with 90% identity at the amino acid level and almost perfect conservation between the functional domains. Furthermore, as in the human gene, the 3' end of the Cacna1f gene maps within 5 kb of the 5' end of the mouse synaptophysin gene in a region orthologous to Xp11.23. Using in situ hybridization, Cacna1f was found to be expressed in the inner and outer nuclear layers and the ganglion cell layer of the retina.

Retro-recombination screening of a mouse embryonic stem cell genomic library.

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for 'rolling out' plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage-plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed 'retro-recombination screening' (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a lambdaTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage lambda into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.

ES cell neural differentiation reveals a substantial number of novel ESTs.

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease.

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination.

Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.

DNA-dependent protein kinase acts upstream of p53 in response to DNA damage.

The tumour suppressor p53 becomes activated as a transcription factor in response to DNA damage, but the mechanism for this activation is unclear. A good candidate for an upstream activator of p53 is the DNA-dependent protein kinase (DNA-PK) that depends on the presence of DNA breaks for its activity. Here we investigate the link between DNA damage and the activation of DNA-PK and of p53. To determine whether DNA-PK is an upstream mediator of the p53 DNA-damage response, we analysed a severe combined-immunodeficiency (SCID) mouse cell line, SCGR11, and the human glioma cell line M059J . Both cell lines lack any detectable DNA-PK activity. We find that p53 is incapable of binding to DNA in the absence of DNA-PK, that DNA-PK is necessary but not sufficient for activation of p53 sequence-specific DNA binding, and that this activation occurs in response to DNA damage. Our results establish DNA-PK as a link between DNA damage and p53 activation, and reveal the existence of a mammalian DNA-damage-response pathway.

Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination.

Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.

Murine subtilisin-like proteinase SPC6 is expressed during embryonic implantation, somitogenesis, and skeletal formation.

During mammalian embryogenesis, proteinases are important for both matrix remodeling and the activation of latent growth factors. As subtilisin-like prohormone convertases (SPCs) have recently been found to activate members of the matrix metalloproteinase and transforming growth factor-beta (TGF-beta) families, we sought to investigate the role of this gene family in murine implantation and embryogenesis. Using active site polymerase chain reaction (PCR) cloning, four members of the SPC family were identified at embryonic day 6.5: SPC1, SPC2, SPC3, and SPC6. In situ hybridization analysis of sectioned E6.5 embryos in utero demonstrated strong SPC6 expression in differentiated decidua, overlapping and extending beyond the region previously described for the metalloproteinase inhibitor TIMP-2. Lower levels of SPC6 expression were observed in trophoblasts and in the ectoplacental cone, suggesting multiple roles for this enzyme in implantation. Northern analysis showed that SPC6 mRNA in embryos is represented by two distinct sizes of message--the isoform SPC6-1 (3.0 kb) is most abundant at all stages, but significant levels of SPC6-b (6.0 kb) occur in E12.5 embryos. Whole mount in situ hybridization to E8.5 embryos demonstrated strong SPC6 expression in the most posterior somite. This somitic staining moved caudally with the developing embryo and by E10.5 became localized to the posterior of the tail, indicating that SPC6 is involved in somitogenesis. SPC6 was expressed at low levels throughout the embryo, except in the developing nervous system, and strong expression was observed in the first branchial arch and in skeletal regions of the developing vertebrae, limbs, and craniofacium, suggesting additional roles for SPC6 in skeletogenesis.

A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains.

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/BIN, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.