Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI

Abstract

Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES-derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage-specific embryonic antigen-4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm-, mesoderm-, and endodermderivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue-specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity

“…Cynomolgus monkey ES cell lines were established, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficiency mice as described previously (10). Undifferentiated ES cells were maintained on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts in DMEM͞F-12 supplemented with 0.1 mM 2-mercaptoethanol͞1,000 units/ml leukemia inhibitory factor͞20% knockout serum replacement (GIBCO)͞4 ng/ml basic fibroblast growth factor.…”

“…Undifferentiated ES cells were maintained on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts in DMEM͞F-12 supplemented with 0.1 mM 2-mercaptoethanol͞1,000 units/ml leukemia inhibitory factor͞20% knockout serum replacement (GIBCO)͞4 ng/ml basic fibroblast growth factor. Subculturing of ES cells was performed by using 0.25% trypsin in PBS with 20% knockout serum replacement and 1 mM CaCl 2 as described (10).…”

“…Considering the usefulness in medical research, we have recently established pluripotent embryonic stem cell lines from blastocysts of Macaca fascicularis (cynomolgus monkey), which is commonly used in preclinical studies (10). ES cells derived from primate (human and monkey) blastocysts possess a number of characteristics distinct from mouse cells, such as surface antigens, leukemia inhibitory factor-independency, and long doubling times (10)(11)(12)(13)(14).…”

“…ES cells derived from primate (human and monkey) blastocysts possess a number of characteristics distinct from mouse cells, such as surface antigens, leukemia inhibitory factor-independency, and long doubling times (10)(11)(12)(13)(14). In this study, we have tested the SDIA method with primate ES cells and investigated its potential application for producing medically useful cells.…”

Efficient and stable Sendai virus-mediated gene transfer into primate embryonic stem cells with pluripotency preserved

“…18 The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 15% ES cell-qualified fetal calf serum (FCS; Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, St Louis, MO, USA), 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin, Irvine Scientific, Santa Ana, CA, USA). The ES cell colonies were routinely passaged every 3-4 days after dissociation with a combined approach of 0.25% trypsin (Invitrogen) digestion and mechanical cutting.…”