extraction-labeling-purification-lineage-specific-cells-from-human

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

Weill Cornell Medical College

The activation, growth, and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cell types within the ovary; yet, the mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We use bulk and single-cell RNA sequencing to distinguish the transcriptional signature of prospectively isolated granulosa and theca/stroma cell subsets within human antral follicles derived from xenografts or ovaries. The analysis deconstructs phenotypic diversification within small (<4 mm) antral follicles, identifying secreted factors that are differentially enriched between mural and oophorus granulosa cells, and segregating stromal/support and steroidal activity between theca externa and interna, respectively. Multiple factors are differentially expressed in follicles of xenograft versus ovarian origin. These data capture a high-resolution transcriptional signature of granulosa and theca subpopulations and provide a systems-level portrait of cellular diversification in early antral human follicles.

Comparison of Human Antral Follicles of Xenograft versus Ovarian Origin Reveals Disparate Molecular Signatures.

More than 200 live births have been achieved using auto-transplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen-thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3±4.6). Xenografts were recovered across a broad range of intervals (1 to 52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical auto-transplantation.

Eleni Kallinos

Center for Reproductive Medicine and Infertility

Weill Cornell Medical College

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

Eleni Kallinos

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Weill Cornell Medical College

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

Center for Reproductive Medicine and Infertility