Artificial oocytes through haploidization of mouse endometrial stromal cells and bone marrow-derived mesenchymal stem cells.
Academic Article
Overview
abstract
OBJECTIVE: To evaluate the feasibility of using endometrial stromal cells (EmSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) as alternative sources of donor nuclei for somatic cell haploidization. DESIGN: To perform somatic cell haploidization, mouse MII oocytes were enucleated and injected with the nucleus of cumulus cells (CCs), EMSCs or BM-MSCs. Intact MII oocytes served as controls. Oocytes following haploidization and control were inseminated and cultured up to 96 hours in a time-lapse incubator to assessed embryo development and morphokinesis. Blastocysts were either cryopreserved for future genetic analysis or transferred to surrogate pseudo-pregnant mice. SUBJECTS: Female B2D2F1 mice (ooplasm donor), Male B6-EGFP mice (spermatozoa donor), CD-1 female mice (surrogate) EXPOSURE: Enucleated oocytes underwent somatic cell nuclear transfer using nucleus of CCs, EMSCs or BM-MSCs to generate functional oocytes. These oocytes are fertilized to generate conceptuses. MAIN OUTCOME MEASURES: The primary outcome compared embryo development in the two experimental groups versus control. The secondary outcome was embryo morphokinetics evaluated with time-lapse microscopy. RESULTS: A total of 811 oocytes were enucleated, with a survival rate of 97.5%. SCNT was performed using CCs (n=90), EmSCs (n=394), or BM-MSCs (n=327) resulting in comparable somatic cell fusion rates of 95.2%, 96.0%, and 96.5%, respectively (P=0.53). Comparing the different SCNT groups, the CC cohort had a higher fertilization rate (45.6%) than the EmSC (30.8%, P<0.05) and BM-MSC (26.5%, P<.01) cohorts. However, subsequent embryo development showed significant attrition in the CC cohort where only 14.4% of CC embryos developed into blastocysts compared to 25.6% of EmSC embryos (P=0.04) and 19.6% of BM-MSC embryos (P=0.29). In terms of embryo morphokinetics, all SCNT groups had slower embryo progression (P<0.01) than the control group. However, EmSC and BM-MSC embryo development was faster than CC embryos from syngamy to the 8-cell stage (48.8 vs. 48.5 vs. 58.5 h, respectively, P<0.01). All EmSC embryos and eight out of nine BM-MSC embryos displayed heterozygosity, confirming biparental contribution from the somatic cell and sperm genome. In the EmSC cohort, 42 blastocysts were transferred, yielding 3 healthy mouse pups (2 male, 1 female). All 3 pups displayed biparental inheritance, grew to adulthood and produced 3 healthy first-generation litters. CONCLUSION: Somatic cell haploidization of EmSCs and BM-MSCs generated oocytes capable of full pre-implantation development and yielded healthy offspring. The utilization of genotyped donor nuclei for neogametogenesis may represent a feasible fertility treatment option for individuals with complete absence of oocytes.
