Biomedical subjects

Paul A De Sousa

Publications and source records attributed to Paul A De Sousa.

7 recordsLinked to original sources

PubMed2006

Maintenance of pregnancy in pigs with limited viable embryos.

Interest is increasing in assisted reproductive technologies in the pig involving embryo cryopreservation, cloning, and genetic modification. Although inherently inefficient and variable in their outcome, the successful application of these techniques in this species is confounded by unique mechanisms for pregnancy recognition and maintenance that require a minimum number of viable embryos during a critical window of time early in gestation. These mechanisms require both local and systemic interactions between conceptuses and the maternal reproductive axis. Here, we describe a method wherein cotransfer of parthenogenetic embryos with the capacity for limited development can be used to sustain the pregnancy of fewer than four viable conceptuses to term.

Animals↗

PubMed2006

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines.

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.

Animals↗

PubMed2006

Optimising the therapeutic safety of human embryonic stem cells through the evolution of defined culture systems supporting their isolation.

Realising the therapeutic promise of embryo-derived stem cells is absolutely dependent on the evolution of defined and quality assured in vitro culture systems to mediate their isolation, expansion and delivery. Critical to the safety of these systems, both for the transplant recipient and the general population, is the necessity to eliminate dependence on animal cell-derived reagents to reduce the risk of cross-specific disease transmission. This paper discusses progress in the development of defined culture systems for human embryonic stem cell isolation and the challenges that still must be overcome.

Animals↗

PubMed2006

The road to providing human embryo stem cells for therapeutic use: the UK experience.

Harnessing the unparalleled properties of human embryo stem cells (hESCs) for the therapeutic treatment of disease and injury will require a convergence of scientific developments with regulatory standards. In the case of the latter, it is especially critical that standards for clinically assisted reproduction be harmonized with those governing human cell and tissue transplantation, most notably with respect to procurement, donation, testing, processing, preservation, storage and distribution of cells. In the UK, existing infrastructure to address these considerations is undergoing extensive reorganization to keep pace with evolving European Union standards. The present best paradigm for defining standards for the therapeutic use of embryo-derived stem cells is experience with adult haematopoietic stem cells (HSC). However, compared with adult-derived stem cell, the origin of embryo-derived stem cells from limiting quantities of tissue and their absolute dependence on in vitro culture to realise their therapeutic potential, makes optimization of their isolation and cultivation of even greater importance. Most notable is the requirement to create animal cell product-free culture environments to reduce the risk of cross-specific disease transmission. In the present paper, we review present and emerging standards in the isolation and banking of human embryo-derived stem cells for therapeutic use in the UK and international progress in the development of defined culture systems for this purpose.

Biological Specimen Banks↗

PubMed2003

Human cloning: can it be made safe?

There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

Animals↗

PubMed2002

Improvement of an electrical activation protocol for porcine oocytes.

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

Amino Acids↗

PubMed2002

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy.

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

Animals↗