BIOMEDICAL ETHICS SEMINAR Embryonic Stem Cell Chimera Research: What is it? What are its benefits? What concerns does it raise? Mark Mercola, Ph.D. Professor, Stem Cell and Regeneration Program, The Burnham Institute, and Department of Pathology (adjunct), UCSD School of Medicine March. 17, 2004

…the sovereignty of man lieth hid in knowledge…

Heritage of Experimental Biology Leading to Research Using Chimeric Embryos 17th-18th centuries: 17th-18th centuries: Initial experiments challenging spontaneous generation and demonstration that sperm activate eggs (Redi, Spallanzani) Initial experiments challenging spontaneous generation and demonstration that sperm activate eggs (Redi, Spallanzani) Regeneration in amphibians, worms, snails (Spallanzani, Tremblay) Regeneration in amphibians, worms, snails (Spallanzani, Tremblay) 19th and early 20th Centuries: 19th and early 20th Centuries: Concept of a germ cell as set aside distinct from somatic cell (August Weismann) Concept of a germ cell as set aside distinct from somatic cell (August Weismann) Wilhelm Roux: hot needle killing one blastomere of frog embryo -> mosaic embryos Wilhelm Roux: hot needle killing one blastomere of frog embryo -> mosaic embryos Driesch, isolation of sea urchin blastomeres and McClendon, isolated frog blastomeres -> regulative development Driesch, isolation of sea urchin blastomeres and McClendon, isolated frog blastomeres -> regulative development Horstadius, Speman: inductive interactions between blastomeres Horstadius, Speman: inductive interactions between blastomeres

Hans Spemann and Hilde Mangold Organizer experiment Organizer experiment Transplantation of cells from one (pigmented) to another (unpigmented) embryo Transplantation of cells from one (pigmented) to another (unpigmented) embryo Evaluated ability of transplanted tissue to induce/organize structures in host tissue Evaluated ability of transplanted tissue to induce/organize structures in host tissue

Potency of Individual Cells in Mammalian Embryos Potency tested by dispersion and reaggregation with different embryo to make chimeric embryos, tracking the donor cells within the host embryo Potency tested by dispersion and reaggregation with different embryo to make chimeric embryos, tracking the donor cells within the host embryo Totipotency of individual cells probably persists until implantation Totipotency of individual cells probably persists until implantation Nuclear potency persists longer, perhaps in many somatic nuclei remain totipotent or can be “re-booted”. Nuclear potency persists longer, perhaps in many somatic nuclei remain totipotent or can be “re-booted”.

Embryonic Stem Cells Derived from pre- implantation embryos; cultures of inner cell mass cells maintained in a totipotent state in culture Derived from pre- implantation embryos; cultures of inner cell mass cells maintained in a totipotent state in culture Totipotency demonstrated by chimeras Totipotency demonstrated by chimeras Regenerative potential is only beginning to be explored and requires understanding how to direct differentiation to target tissues Regenerative potential is only beginning to be explored and requires understanding how to direct differentiation to target tissues

Spontaneous Cardiomyocyte Formation in Aggregate Cultures of hES Cells How to channel this ability to differentiate to produce target tissues?

Examples of Tissues for which Interspecies Chimeras have helped define Potency, Fate, and Function Neural crest Neural crest Somites Somites Brain Brain Tumors Tumors These experiments do not elicit much ethical concern

A Few Possible Reasons for Minimal Concern of Grafting Experiments Adult, so no chance of germline transmission Adult, so no chance of germline transmission Non-human host Non-human host No involvement of human CNS function No involvement of human CNS function Degree of mosaicism is poor - e.g. large bits of tissues generally transplanted Degree of mosaicism is poor - e.g. large bits of tissues generally transplanted

Mouse Embryonic Stem Cell-based Chimeras Introduction of ES cells into pre-implantation blastocyst Introduction of ES cells into pre-implantation blastocyst Mix with host cells Mix with host cells Generate high degree of mosaicism Generate high degree of mosaicism Contribute to germline = sperm and egg Contribute to germline = sperm and egg Basis for gene knockout technology, which underlies a substantial national research effort Basis for gene knockout technology, which underlies a substantial national research effort Most people do not voice ethical concerns about this technology Most people do not voice ethical concerns about this technology

Human ES cell lines Derived similarly to mouse ES lines Derived similarly to mouse ES lines Pluripotency is assumed from teratoma assays Pluripotency is assumed from teratoma assays Pluripotency makes them advantageous for tissue regeneration Pluripotency makes them advantageous for tissue regeneration Many people voice ethical concerns Many people voice ethical concerns Restrictions on use with federal funding Restrictions on use with federal funding Relatively few restrictions with private funding Relatively few restrictions with private funding

Human ES Cell Line -Mouse Embryo Chimera Pros and Cons: Pros and Cons: Pro: Test potency of human ES cell Pro: Test potency of human ES cell Pro: Create disease models for research Pro: Create disease models for research Con: Widely available hES cell lines probably will not incorporate effectively Con: Widely available hES cell lines probably will not incorporate effectively Con: Fear that might contribute to germline Con: Fear that might contribute to germline Con: Moderate ethical unease Con: Moderate ethical unease Consider: Similar issues with other cell types, except germline potential; therefore a large debate is looming on horizon How to establish mechanisms to ensure ethical practice at research institutions?

What Makes a Procedure Ethically Challenging? Example 1: Human ES cells into human embryos Example 1: Human ES cells into human embryos Consider hES cells placed into preimplantation embryos, even if to cure a genetic disease? Consider hES cells placed into preimplantation embryos, even if to cure a genetic disease? What if cells are differentiated first, so that they rescue a defect but cannot contribute to germline? What if cells are differentiated first, so that they rescue a defect but cannot contribute to germline? Example 2: Human hematopoietic stem cells into primates to generate animals that could donate blood Example 2: Human hematopoietic stem cells into primates to generate animals that could donate blood What makes people uneasy? What makes people uneasy? How to draw line between ethical and unethical? How to draw line between ethical and unethical? If chimeras are to be permitted, is there a tissue that must remain free from donor cells? If chimeras are to be permitted, is there a tissue that must remain free from donor cells?

Ethical Advisory Board Distinct from animal use and human subject IRB committees Distinct from animal use and human subject IRB committees Consists of lawyers and ethicists Consists of lawyers and ethicists Reduces risk of performing a procedure that might be illegal, unethical or otherwise engender adverse publicity Reduces risk of performing a procedure that might be illegal, unethical or otherwise engender adverse publicity