Hooman Sadri-Ardekani

Propagation of human spermatogonial stem cells in vitro.

CONTEXTnYoung boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility.nnnOBJECTIVEnTo establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation.nnnDESIGN, SETTING, AND PARTICIPANTSnStudy performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation.nnnMAIN OUTCOME MEASURESnPropagation of spermatogonial stem cells over time.nnnRESULTSnTesticular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture.nnnCONCLUSIONnLong-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

In Vitro Propagation of Human Prepubertal Spermatogonial Stem Cells

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

Embryonic stem cell-like cells derived from adult human testis

BACKGROUNDnGiven the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis.nnnMETHODSnTestis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity.nnnRESULTSnIn testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice.nnnCONCLUSIONSnMultipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells

Abstract Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5%% fetal calf serum, about 36%% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70%% decrease in expression of the cell cycle inhibitor CDKN1B (P27kip1) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25%% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70%% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be.

Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study

OBJECTIVEnTo study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system.nnnDESIGNnExperimental laboratory study.nnnSETTINGnReproductive biology laboratory, academic medical center.nnnPATIENT(S)nAcute lymphoblastic leukemia cells from three patients and testicular cells from three other patients.nnnINTERVENTION(S)nAcute lymphoblastic leukemia cells were cultured alone or in combination with testicular cells, at various concentrations, in a system that has recently been developed to propagate human spermatogonial stem cells.nnnMAIN OUTCOME MEASURE(S)nViability of ALL and testicular cells during culture was evaluated by flow cytometry using markers for live/dead cells. Furthermore, the presence of ALL cells among testicular cells was determined by highly sensitive (1:10,000 to 1:100,000 cells) patient-specific antigen-receptor minimal residual disease polymerase chain reaction. The presence of spermatogonia at the end of culture was determined by reverse transcription-polymerase chain reaction for ZBTB16, UCHL1, and GPR125.nnnRESULT(S)nThe ALL cells cultured separately did not survive beyond 14 days of culture. When cultured together with testicular cells, even at extremely high initial concentrations (40% ALL cells), ALL cells were undetectable beyond 26 days of culture. Reverse transcription-polymerase chain reaction confirmed the presence of spermatogonia at the end of the culture period.nnnCONCLUSION(S)nOur pilot study shows that the described testicular cell culture system not only allows for efficient propagation of spermatogonial stem cells but also eliminates contaminating ALL cells.

Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture

OBJECTIVEnTo determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture.nnnDESIGNnExperimental basic science study.nnnSETTINGnReproductive biology laboratory.nnnPATIENT(S)nCryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nTesticular cells before and 50xa0days after culturing were subjected to ITGA6 magnetic-activated cell sorting to enrich for SSCs. Individual spermatogonia were analyzed for aneuploidies with the use of single-cell 24-chromosome screening. Furthermore, the DNA methylation statuses of the paternally imprinted genes H19, H19-DMR (differentially methylated region), and MEG3 and the maternally imprinted genes KCNQ1OT1 and PEG3 were identified by means of bisulfite sequencing.nnnRESULTS(S)nAneuploidy screening showed euploidy with no chromosomal abnormalities in all cultured and most noncultured spermatogonia from both patients. The methylation assays demonstrated demethylation of the paternally imprinted genes H19, H19-DMR, and MEG3 of 11%-28%, 43%-68%, and 18%-26%, respectively, and increased methylation of the maternally imprinted genes PEG 3 and KCNQ1OT of 13%-50% and 30%-38%, respectively, during culture.nnnCONCLUSION(S)nIn the current culture system for human SSCs propagation, genomic stability is preserved, which is important for future clinical use. Whether the observed changes in methylation status have consequences on functionality of SSCs or health of offspring derived from transplanted SSCs requires further investigation.

Parental attitudes toward fertility preservation in boys with cancer: context of different risk levels of infertility and success rates of fertility restoration

OBJECTIVEnTo measure the parental attitudes toward fertility preservation in boys with cancer.nnnDESIGNnRetrospective cohort study.nnnSETTINGnQuestionnaire survey via regular mail.nnnPATIENT(S)nA total of 465 families whose sons were already treated for cancer.nnnINTERVENTION(S)nThe questionnaire was designed for two groups based on childs age at the time of cancer diagnosis: <12 and ≥12 years old.nnnMAIN OUTCOME MEASURE(S)nDescriptive statistics regarding a positive or negative attitude of parents toward fertility preservation options in the context of different risk levels of infertility and success rates of fertility restoration.nnnRESULT(S)nThe response rate was 78%. Sixty-four percent of parents of boys ≥12 years old would agree to store sperm obtained by masturbation and/or electroejaculation, and 54% of parents of boys <12 years old would agree to store a testicular biopsy. If the risk of infertility or the success rate of fertility restoration were ≤20%, more than one-fourth of parents would still opt for fertility preservation.nnnCONCLUSION(S)nAll parents should be counseled about the risks of infertility due to cancer treatment, because many parents want to preserve their sons fertility even if the risk of becoming infertile or the chances on fertility restoration are low.