Itai Gat1, Leila Maghen2, Melissa Filice2, Brandon Wyse2, Khaled Zohni3, Keith Jarvi4, Kirk C Lo4, Andrée Gauthier Fisher2, Clifford Librach5. 1. Create Fertility Centre, Toronto, Ontario, Canada; Pinchas Borenstein Talpiot Medical Leadership Program, Sheba Medical Center, Ramat Gan, Israel; Sackler school of medicine, Tel Aviv university, Tel Aviv, Israel. 2. Create Fertility Centre, Toronto, Ontario, Canada. 3. Create Fertility Centre, Toronto, Ontario, Canada; Department of Reproductive Health and Family Planning, National Research Center, Cairo, Egypt. 4. Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada. 5. Create Fertility Centre, Toronto, Ontario, Canada; Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada; Department of Obstetrics and Gynecology, Women's College Hospital, Toronto, Ontario, Canada. Electronic address: drlibrach@createivf.com.
Abstract
OBJECTIVE: To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells. DESIGN: Basic science study. SETTING: Urology clinic and stem cell research laboratory. PATIENT(S): Eight human testicular samples. INTERVENTIONS(S): Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12 days. MAIN OUTCOME MEASURE(S): TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates. RESULT(S): TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8 h and 110.5 ± 68.3 h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively). CONCLUSION(S): Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports.
OBJECTIVE: To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells. DESIGN: Basic science study. SETTING: Urology clinic and stem cell research laboratory. PATIENT(S): Eight human testicular samples. INTERVENTIONS(S): Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12 days. MAIN OUTCOME MEASURE(S): TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates. RESULT(S): TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8 h and 110.5 ± 68.3 h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively). CONCLUSION(S): Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports.
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