In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities

Abstract

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Induction of Meiosis from Human Pluripotent Stem Cells

“…We additionally tested mTeSR1 (which maintains primed pluripotency), HENSM (which induces naïve pluripotency) ( 36 ), StemPro-34 based spermatogonial stem cell culture medium ( 37 ), and APEL2 (Stemcell Technologies; a “neutral” medium lacking growth factors).…”

Sex chromosome aneuploidies and fertility: 47,XXY, 47,XYY, 47,XXX and 45,X/47,XXX

“…As a proof of concept, Galdon and colleagues isolated testicular cells from three biopsies from adolescents with KS. A heterogeneous mix of SSCs and somatic cells was cultured ( 85 ). After a few days, qPCR analysis revealed characteristic gene expression from undifferentiated spermatogonia, Leydig, Sertoli, and peritubular cells with at least 1 × 10 6 -fold increase in cell number.…”

“…As time in culture increased, in addition to XXY cells, XY and XX cells were identified. The investigators considered such expansion could potentially enable SSC transplantation ( 85 ). To date, there is no specific technique to return the cultured cells to the recipient, although in animals direct microinjection to the seminiferous tubules or the rete testis has been accomplished ( 82 ).…”

The correlation between clinical features and ultrastructure of testis of non-mosaic Klinefelter's syndrome patients with hypogonadism and androgen deficiency: A case report

“…The usually karyotype presenting is non-mosaic 47 XXY (80%–85%), and other karyotypes include 46, XY/47, XXY chimeric type, 48, XXXY, 49, XXXXY, etc. [ 2 ]. Here, we report a case of KS patients with hypogonadism and androgen deficiency, whose testicular ultrastructure showed hyaline convoluted tubules, vacuolated Sertoli cells and Leydig cells.…”