Nonhuman primate parthenogenetic stem cells

doi:10.1073/pnas.2034195100

Nonhuman primate parthenogenetic stem cells

^*Center for Neurobehavioral Study of Alcohol, Department of Physiology and Pharmacology and ^‡Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157; ^§Department of Microbiology and Immunology, Mayo Clinic, Rochester, MN 55905; ^¶Sloan-Kettering Cancer Center, New York, NY 10021; ^**Advanced Cell Technology, Worcester, MA 01605; ^∥Millennium Pharmaceuticals, Cambridge, MA 02139; and ^††Department of Animal Science-Physiology, Michigan State University, East Lansing, MI 48824

Abstract

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describe in vitro parthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

Footnotes

↵ † To whom correspondence should be addressed. E-mail: kvrana{at}wfubmc.edu or cibelli{at}msu.edu.
This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Regenerative Medicine,” held October 18-22, 2002, at the Arnold and Mabel Beckman Center of the National Academies of Science and Engineering in Irvine, CA.
Abbreviations: ES cell, embryonic stem cell; Cyno-1 cell, parthenogenetically derived stem cell line from the cynomolgus macaque; bFGF, basic fibroblast growth factor; Snrpn, small nuclear ribonucleoprotein polypeptide N; TUJ1, β-tubulin III; PBL, peripheral blood lymphocytes; ICM, inner cell mass.