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Biomedical subjects

Steven L Stice

Publications and source records attributed to Steven L Stice.

At least 19 recordsLinked to original sources
PubMed2006

Production of cloned calves using roscovitine-treated adult somatic cells as donors.

The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize adult bovine granulosa cells in the G0/G1 cell cycle stage. The resulting fetal and calf survival after transfer of cloned embryos was enhanced in the roscovitine-treated group compared with serum-starved controls. The methods described in this chapter outline (1) the preparation of donor cells, (2) the preparation of recipient oocytes, and (3) the production of cloned embryos. The first section involves methods for the preparation of donor cell stocks from isolated granulosa cells and the roscovitine treatment of the cells before nuclear transfer. The second section explains procedures of in vitro maturation of recipient oocytes. The last section involves methods for the production of cell-oocyte complexes, the fusion of the complexes, and the activation, in vitro culture, and transfer into recipient females of cloned embryos.

Animals↗
PubMed2006

Human embryonic stem cells: challenges and opportunities.

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

Animals↗
PubMed2006

Uniform adherent neural progenitor populations from rhesus embryonic stem cells.

Rhesus and human embryonic stem cells (ESCs) are similar, making rhesus ESCs an appropriate preclinical allograft model for refining stem cell therapies. Use of rhesus ESC-derived neural progenitors (NPs) in preclinical applications will be enhanced if the neural derivation process is scalable and free from contaminating ESCs or nonneural cells. In this study, we have quantified temporal gene expression changes of rhesus ESC differentiated to uniform NPs using simple feeder-free adherent cultures. NPs exhibited a significant up-regulation of neural-specific genes and a downregulation of pluripotency genes. Additionally, expression of Hu, MAP2, and Tuj1, shows that NPs can form post-mitotic neurons. This study represents a simple and scalable means of producing adherent primate NPs for preclinical testing of neural cell-based therapy.

Animals↗
PubMed2006

In vitro derivation and expansion of endothelial cells from embryonic stem cells.

Vascular endothelial cells or endothelial progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications, including cell-based therapies and tissue engineering. Embryonic stem (ES) cells serve as an excellent in vitro system for studying differentiation events and for developing methods of generating various specialized cells for future regenerative therapeutic applications. Two obstacles associated with using embryonic stem cells include (1) isolating homogeneous populations of differentiated cells and (2) obtaining terminally differentiated cell populations that are capable of proliferating further. Here, we describe methods for isolating purified proliferating populations of endothelial cells from mouse ES cells using Flk-1-positive cells, vascular endothelial growth factor supplementation, and a highly selective manual selection technique. This methodology, although rigorous, overcomes two current obstacles in ES derivation and culture by generating highly purified (>96%) populations of actively proliferating endothelial cells from mouse ES cells. Using this in vitro derivation procedure, millions of cells at various stages of differentiation may be obtained and expanded up to 25 population doublings.

Animals↗
PubMed2005

Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.

Animals↗
PubMed2005

Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions.

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.

Animals↗
PubMed2005

Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor.

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.

Adenoviridae↗
PubMed2005

Human motor neuron differentiation from human embryonic stem cells.

The therapeutic potential of embryonic stem (ES) cells is promising, but in many cases limited by our inability to promote their differentiation to specific cell types, such as motor neurons. Here we provide the first report of the successful differentiation of human ES cells to cells of a motor neuron phenotype. A renewable source of neuroepithelial cells was generated from human ES cells. Extracellular signals were then employed to induce motor neuron differentiation and related gene expression by these cells. OLIG2 and HLXB9 gene expression increased upon the addition of basic fibroblast growth factor, retinoic acid, and sonic hedgehog, as a motor neuron phenotype expressing Islet1 and choline acetyltransferase (ChAT) developed. This study demonstrates that neuroepithelial cells derived from human ES cells are renewable progenitors capable of generating motor neurons at levels that may be therapeutically useful. Sonic hedgehog, basic fibroblast growth factor, and retinoic acid differentially influence human motor neuron differentiation by mechanisms that remain to be defined.

Cell Culture Techniques↗
PubMed2004

Comparative transcriptional profiling of two human embryonic stem cell lines.

Human embryonic stem cells (ESCs) have generated enormous interest due to their ability to self-renew and produce many different cell types. In conjunction with microarray technology, human ESCs provide a powerful tool for employing a systems-based approach to deciphering the molecular mechanisms that control pluripotency and early development. Recent work has focused on defining "stemness" and pluripotency based on different experimental and analytical approaches in both mouse and human ESCs. Using a mixed linear model statistical approach, we report a stringent direct comparison between data sets obtained from two human ESCs (BG01 and H1) in order to obtain a list of genes that are enriched in ESCs. In addition, we used another pluripotent population derived from BG01 ESCs to obtain a list of genes that we consider important to the maintenance of pluripotency. A total of 133 genes overlapped between the three pluripotent populations. A majority of the 133 genes were classified under the key functional categories of cell-cycle regulation, signaling, and regulation of transcription. Key genes expressed were Oct4, Sox2, LeftyA, and Fgf2. Also found to be enriched in all three populations is FLJ10713, a gene encoding a hypothetical protein of unknown function that has been shown in earlier studies to possess a homolog in mouse ESCs and also to cluster tightly with Oct4 in human ESCs. Although there were many genes unique to each pluripotent population, they shared similarities based on functional ontologies that define pluripotency. The significance of our studies underscores the need for direct comparison of stem cell populations that share biological similarities using uniform stringent analytical approaches, in order to better define pluripotency. Our findings have important implications for the maintenance of pluripotency and in developing directed differentiation strategies for various regenerative applications.

Algorithms↗
PubMed2004

Transcriptional profiling of initial differentiation events in human embryonic stem cells.

Currently, there are no differentiation strategies for human embryonic stem cells (hESCs) that efficiently produce one specific cell type, possibly because of lack of understanding of the genes that control signaling events prior to overt differentiation. sed HepG2 cell conditioned medium (MEDII), which induces early differentiation in mouse ES cells while retaining pluripotent markers, to query gene expression in hESCs. Treatment of adherent hESCs with 50% MEDII medium effected differentiation to a cell type with gene expression similar to primitive streak stage cells of mouse embryos. MEDII treatment up-regulates TDGF1 (Cripto), a gene essential for anterior-posterior axis and mesoderm formation in mouse embryos and a key component of the TGFB1/NODAL signaling pathway. LEFTYA, an antagonist of NODAL/TDGF1 signaling expressed in anterior visceral endoderm, is down-regulated with MEDII treatment, as is FST, an inhibitor of mesoderm induction via the related INHBE1 pathway. In summary, the TGFB1/NODAL pathway is important for primitive-streak and mesoderm formation and in using MEDII, we present a means for generating an in vitro cell population that maintains pluripotent gene expression (POU5F1, NANOG) and SSEA-4 markers while regulating genes in the TGFB1/NODAL pathway, which may lead to more uniform formation of mesoderm in vitro.

Carcinoma, Hepatocellular↗
PubMed2004

Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events.

Embryonic stem cells are characterized by their ability to propagate indefinitely in culture, maintaining a normal karyotype and their undifferentiated state. They have the potential of differentiating into any specialized cell type in the body. An understanding of the transcriptional profile related to pluripotency and early development is necessary to better tap their developmental potential and also maintain their undifferentiated phenotype. Currently, several techniques are in use to ascertain the gene expression profile of embryonic stem cells. This review summarizes the information generated using microarray and other approaches on the gene expression analyses of stem cells in both mouse and human cell lines. We also discuss specific approaches useful in future studies aimed at further deciphering the pluripotent nature of human embryonic stem cells.

Animals↗
PubMed2003

Generation of bovine transgenics using somatic cell nuclear transfer.

The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

Animals↗
PubMed2003

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons.

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.

Action Potentials↗
PubMed2003

Gene expression and in vitro development of inter-species nuclear transfer embryos.

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.

Animals↗
PubMed2003

Purified and proliferating endothelial cells derived and expanded in vitro from embryonic stem cells.

Embryonic stem (ES) cells serve as an excellent in vitro system for studying differentiation events and for developing methods of generating various specialized cells for future regenerative therapeutic applications. Two obstacles associated with using embryonic stem cells include (a) isolating homogeneous populations of differentiated cells and (b) obtaining terminally differentiated cell populations that are capable of proliferating further. Here, the authors describe methods in which they have overcome these two obstacles by generating highly purified populations (>96%) of actively proliferating endothelial cells from mouse ES cells. Briefly, 60,000 ES cells progress through three different stages of cell induction/expansion and two cell isolation procedures, generating over 300 million endothelial cells. These ES-derived endothelial cells display characteristics similar to vascular endothelial cells in that they express several common endothelial markers, they form two-dimensional (2D) tubelike structures as well as complex microvessels in three-dimensional (3D) collagen type I gels, and they retain the ability to reorganize their cytoskeleton in response to mechanical forces. Our findings indicate that it is possible to obtain proliferating populations of homogeneous endothelial cells from mouse ES cells without genetically manipulating the ES cells or coculturing with feeder cells.

Animals↗
PubMed2002

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells.

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.

Animals↗
PubMed2002

In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype.

This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 x 10(5) cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P < 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.

Animals↗