191 IN VIVO AND IN VITRO DIFFERENTIATION OF EMBRYONIC STEM CELLS DERIVED FROM PARTHENOGENETIC EMBRYOS IN MICE

Abstract

Availability of embryonic stem (ES) cells opens the prospect for regenerative medicine. However, ES cells genetically mismatched to diseased individuals cause immunological rejection. In this study, we established ES cells from parthenogenetic embryos in mice and examined their pluripotency. Oocytes were collected from (C57BL/6xDBA)F1 mice (BDF1) by superovulation. Parthenogenetic diploid embryos were produced by activation treatment in 5 mM SrCl₂ in Ca²⁺-free KSOM medium for 2 h, followed by cultivation in 5 μg mL⁻¹ cytochalasin B for 6 h. The zonae pellucidae of embryos developed to the blastocyst stage in vitro were removed by a 5-min incubation in 0.5% pronase. Inner cell masses (ICMs) isolated immunosurgically were seeded on the feeder layers (mitomycin C-treated mouse embryonic fibroblasts) in DMEM supplemented with 15% Knock-Out Serum Replacement (Invitrogen), 2 mM L-glutamine, non-essential amino acids, β-mercaptoethanol and 10³ U mL⁻¹ of Leukemia inhibitory factor (LIF) at 37°C in a humidified atmosphere with 5% CO₂ in air. The attached ICM cells were mechanically disaggregated and seeded on the fresh feeder layers. After several passages, parthenogenetic ES (PnES) cell lines were established. The efficacy of establishing PnES cell lines was 66% (37/56). To examine the characteristics of PnES cell lines, seven lines were subjected to histochemical and immunohistochemical analysis. All showed alkaline phosphatase activity and immunoreactivity to anti-SSEA-1 and anti-Oct4 antibodies. They maintained euploid sets of choromosomes at 29; 59%. PnES cells from two of the seven lines were injected into 59 host blastocysts obtained from ICR mice, resulting in 16 chimeric offspring (27%). In another experiment, injection of ICM cells and ES cells obtained from fertilized BDF1 blastocysts and ICM cells obtained from BDF1 parthenogenetic blastocysts also produced chimeric offspring (35%, 7/20; 46%, 6/13; and 53%, 10/19, respectively). However, no chimeric mouse with germline transmission was obtained from PnES cells. Injection of 1 × 10⁷ of PnES cells into SCID mice formed teratocarcinomas. Immunohistochemical analysis showed cells positive for nestin (specific to neuroepitherial stem cells), Tu-J (class III β-tublin), NF-M (neurofilament), desmin (muscle), and albumin (hepatocytes), which indicated their differentiation potency to the cells derived from all three germ layers. Simple embryoid bodies produced from these cell lines were plated on tissue culture dishes under conditions for induction of differentiation. Immunohistochemistry and RT-PCR analysis showed their differentiation into neurons (NF-M, nestin), cardiomyocytes and hepato-like cells (albumin, α-fetoprotein). Our results indicate that PnES cells are pluripotent similar to the ES cells from fertilized embryos except for germline transmission and should be tested in cell replacement animal models.