Artificial oocytes through haploidization of mouse endometrial stromal cells and bone marrow-derived mesenchymal stem cells.

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 metaphase II oocytes were enucleated and injected with the nucleus of cumulus cells (CCs), EmSCs or BM-MSCs. Intact metaphase II oocytes served as controls. Oocytes after haploidization and controls were inseminated and cultured up to 96 hours in a time-lapse incubator to assess 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 the 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 experimental groups vs. 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%. Somatic cell nuclear transfer (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. Comparing the different SCNT groups, the CC cohort had a higher fertilization rate (45.6%) than the EmSC (30.8%) and BM-MSC (26.5%) cohorts. However, subsequent embryo development showed significant attrition in the CC cohort, where only 14.4% of CC embryos developed into blastocysts compared with 25.6% of EmSC embryos and 19.6% of BM-MSC embryos. In terms of embryo morphokinetics, all SCNT groups had slower embryo progression 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 hours, respectively). 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 three healthy mouse pups (2 male, 1 female). All three pups displayed biparental inheritance, grew to adulthood and produced three healthy first-generation litters.

CONCLUSION

Somatic cell haploidization of EmSCs and BM-MSCs generated oocytes capable of full preimplantation 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.