From Cell to Production

Technical Challenges of Cloning Pigs for BioMedical Research

Somatic Cell Nuclear Transfer in Mammals

SATACs and Transgenesis

Concerns About Gene Transfer and Nuclear Transfer in Domestic Animals

ES Cells Make Neurons in a Dish

Nuclear Transfer and Gene Targeting in Domestic Animals: Bioreactors of the Future

Application of Nuclear Transfer Technology in the Generation of Pigs for Xenetransplantation

Genomics: Delivering Cell Culture Systems for Tissue Therapy

Nuclear Transfer Technology

Gene Targeting in Domestic Species: Challenges and Opportunities

Homologous Recombination and Genetic Engineering of Transgenic Recombinant Animals

Nuclear Transplantation in the Cow: Future Challenges

Enhancing Transgenics through Cloning

ES Cells Offer is a Power Tool for Understanding the Genetic Control of Tissue Development and for Screening Potential Therapeutic Drugs

Human Germline Engineering -- The Prospects for Commercial Development

Mammalian Artificial Chromosomes for Animal Transgenesis

Understanding Developmental Abnormalities in Offspring Produced by Nuclear Transplantation

Role of Cell Cycle

Cloning and Other Reproductive Technologies for Application in Transgenics

PROSPECTS AND HURDLES IN OPTIMIZING THE VASCULAR SUPPORT OF ENGINEERED TISSUES

Participant:

Robert L. Gendron, Ph.D.
Division of Hematology and Oncology
Childrens Hospital Medical Center
Cincinnati Ohio 45229-3039

One of the major impediments to the successful engineering of replacement tissues for grafting and organ repair is the adequate vascularization of the new tissue. New blood vessels are necessary to support the health and homeostasis of tissue grafts. Moreover, the capillary network supporting engineered tissues could be used to deliver growth factors or drugs to the tissue itself. The identification of molecular switches regulating the formation and growth of blood vessel capillaries (vasculogenesis and angiogenesis) is a necessary step for designing new practical strategies for optimizing graft neovascularization and/or for maintaining the endogenous vascular network in grafted or engineered tissue. Key regulatory molecules controlling the growth and differentiation of capillaries could be targeted using peptidomimetics or exogenous genes to optimize the controlled growth of a supportive blood vessel network in new tissues. However, there is first a need for studying in detail the cells giving rise to capillaries and the genes controlling the signaling pathways making the different endothelia compatible with their destined tissue environments.

Our perspective on this problem has involved an initial focus of generating in vitro model systems of vascular differentiation which are also useful in vivo. We have generated a clonal mouse embryonic endothelial cell line (IEM) by immortalizing the differentiated derivatives of mouse embryonic stem cells. The IEM cells respond to leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) by forming capillary structures in vitro . In vivo, the cytokine treated IEM cells can chimerize developing vascular structures without disrupting development, but do so in a tissue restricted manner (1,2). Recently, Hatzopoulos et al. have also reported that cloned mouse embryonic endothelial cells can integrate normally into specific developing vascular structures (3). Furthermore, the ability of human endothelial progenitor cells to integrate into sites of tissue injury has also recently been reported (4). We surmised that their ability to differentiate into capillaries and to chimerize vascular structures makes IEM a useful model system to isolate and study the effects of genes which might alter the fate of endothelial stem cells in vivo. We are now using the IEM cells for identifying and studying new genes which might be key switches in controlling capillary growth and differentiation. The products of these genes could potentially be utilized to optimize the growth of blood vessel networks in artificially generated or grafted tissues.

We have recently isolated a novel regulatory gene from undifferentiated IEM cells named tubedown-1 (tbdn-1). Transcripts of this gene are expressed highly in undifferentiated endothelial cells and become downregulated as endothelial cells differentiate into capillaries. Tbdn-1 encodes a 700 amino acid protein with motifs predicting a role in chromatin remodeling. Inhibition of tbdn-1 expression by overexpression of antisense cDNA indicates that tbdn-1 may act as a negative regulator of capillary differentiation in IEM cells. If tbdn-1 can alter the growth and differentiation of endothelia in vivo, the tbdn-1 product may be a useful candidate with which to manipulate and remodel capillary networks in artificially engineered tissues. Experiments to test the role tbdn-1 plays in capillary growth in vivo are underway in our laboratory.

Two major technical hurdles slow our current ability to practically manipulate the blood vessel support of organ grafts and engineered tissues. The first is a lack of knowledge of the biology of endothelial stem cells and the differences in endothelia from different tissue sources. What biological attributes make endothelial stem cells developmentally plastic? At what developmental stage should endothelial stem cells be isolated to optimize their ability to integrate into and support engineered tissues? Are endothelial stem cells "generic"; or are there phenotypic differences in endothelia? Our work and the work of others has begun to address some of the features of endothelial progenitor cells but a more complete study of the biology of endothelial stem cells is required to make their successful isolation and consistent usefulness in tissue engineering a reality. The second technical hurdle facing this field is our lack of a complete understanding of the molecular events controlling the growth and differentiation of endothelia. What downstream regulatory proteins control the angiogenic signals stimulated by cytokines such as bFGF and vascular endothelial growth factor (VEGF)? Could such proteins be mimicked or blocked with drugs in order to remodel the vascular tree in a graft? These hurdles will be overcome as we learn more about endothelial stem cells by studying additional endothelial progenitor cell lines and by exploring the properties of the molecules regulating their growth and differentiation. We have isolated a potentially important candidate molecule that may control capillary formation using IEM cells. However, understanding how such molecules fit into the signaling pathways controlling the growth and differentiation of blood vessels requires further study before they can be used practically.

1. Gendron, R.L., Tsai, F.-Y., Paradis, H. and Arceci, R.J. Induction of Embryonic Vasculogenesis by bFGF and LIF in vitro and in vivo. Dev. Biol., 177, 332-347, 1996.

2. Paradis, H., Arceci, R.J., Adams, L. and Gendron, R.L. Differentiation responses of embryonic endothelium to leukemia inhibitory factor. Exp. Cell Res., 240, 7-15, 1998.

3. Hatzopoulos AK, Folkman J, Vasile E, Eiselen GK, and Rosenberg RD. Isolation and characterization of endothelial progenitor cells from mouse embryos. Development 125(8), 1457-1468, 1998.

4. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964-967, 1997.