Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by rapid metastasis and resistance to chemotherapy, properties that are shared by cancer stem cells (CSCs). In pancreatic cancer, tumor cells which possess the properties of CSCs also phenotypically resemble cells that have undergone epithelial-to-mesenchymal transition or EMT. Disabled-2 (Dab2) is a multifunctional scaffold protein frequently downregulated in cancer that has been linked to the process of EMT. However, the role of Dab2 in pancreatic cancer development and progression remains unclear. Downregulation of Dab2 expression in pancreatic cancer cell lines was found to trigger induction of genes characteristic of EMT and the CSC phenotype, while overexpression of Dab2 in the Panc1 cell line blocked the process of TGFβ-stimulated EMT. In addition, selective inhibition of the TGFβRI/RII receptors was found to reverse genes altered by Dab2 downregulation. Dab2 mRNA expression was found to be decreased in PDAC tumor samples, as compared to levels observed in normal pancreatic tissue. Methylation of the Dab2 gene promoter was demonstrated in Stage I PDAC tumors and in the MiaPaCa2 cell line, suggesting that promoter methylation may silence Dab2 expression early in pancreatic cancer progression. These results suggest that Dab2 may function as a tumor suppressor in pancreatic cancer by modulation of the TGFβ-stimulated EMT and CSC phenotype.