Journal: bioRxiv

Article Title: A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells

doi: 10.1101/2021.06.04.447118

Figure Lengend Snippet: Isolation/derivation and characterization of primary SSCs and hSSCs. A) Schematic representing primary SSC isolation: Testicular Sperm Extraction (TESE) biopsies were dissociated into single cells, plated on tissue culture plates, and then 24 hours later the non-adherent germ cells were collected and plated on laminin-coated plates. B) Schematic representing SSC derivation from hiPSCs: hiPSCs are reprogrammed from adult fibroblasts and then cultured with FGF2, GDNF, lipids, and ascorbic acid to promote SSC differentiation. C) hSSC expression of SSC markers GPR125, GFRA1, CD90 and SSEA4, and the pluripotency factor OCT4, and hiPSC expression of OCT4. D) Primary SSC expression of the SSC markers GPR125, GFRA1, CD90 and SSEA4, and the testicular somatic markers ACTA2 and SOX9. All scale bars are 100 μm. Abbrevations: FGF2 = Fibroblast Growth Factor 2, GDNF = Glial Cell-Derived Neurotrophic Factor, GPR125 = G-Protein Coupled Receptor 125, GFRA1 = GDNF Family Receptor Alpha 1, CD90 = Thy-1 Cell Surface Antigen, SSEA4 = Stage-Specific Embryonic Antigen 4, OCT4 = Octamer Binding Protein 4, ACTA2 = Actin Alpha 2, Smooth Muscle, SOX9 = SRY=Box Transcription Factor 9, hiPSC = human induced pluripotent stem cell, SSC = spermatogonial stem cell, hSSC = human induced pluripotent stem cell-derived spermatogonial stem cell, Primary = primary spermatogonial stem cell, TESE = testicular sperm extraction.

Article Snippet: hSSCs were differentiated from hiPSCs as previously described,[ ] with a magnetic activated cell sorting (MACS) step to purify for Thy-1 Cell Surface Antigen (THY1/CD90)-expressing cells. hiPSCs were grown on Matrigel in Minimum Essential Medium Alpha (αMEM, Gibco A10490-01), 1X GlutaMAX (Thermofisher, 35050079), 1X Insulin Transferrin Selenium Liquid Media Supplement (ITS, Millipore Sigma, I13146), 0.2% BSA (Miltenyi Biotec, 130-091-376), 1% penicillin/streptomycin (Sigma Aldrich, P4333), 1 ng/mL human recombinant Fibroblast Growth Factor 2 (FGF2, STEMCELL Technologies, 78003.1), 20 ng/mL animal component-free human recombinant Glial Cell Derived Neurotrophic Factor (GDNF, STEMCELL Technologies, 78139), 0.2% Chemically Defined Lipid Concentrate (Thermofisher, 11905031), and 200 μg/mL L-ascorbic acid (Sigma, A4544).

Techniques: Isolation, Cell Culture, Expressing, Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells

doi: 10.1101/2021.06.04.447118

Figure Lengend Snippet: Survival of hSSCs in xeno-free media conditions. A) Schematic representing the experimental culture conditions: the standard conditions included FBS and BSA supplementation, while the experimental xeno-free conditions had removed FBS and BSA and additional supplementation of PGD-2 and IGF-1. B) Microscope image of the hSSCs in standard conditions. C) Microscope images of the hSSCs in the experimental xeno-free media without IGF-1 or PGD-2, with IGF-1 or PGD-2, and with both IGF-1 and PGD-2. All scale bars are 100 μm. Abbreviations: hSSC = human induced pluripotent stem cell-derived spermatogonial stem cell, FBS = fetal bovine serum, BSA = bovine serum albumin, PGD-2 = prostaglandin D2, IGF-1 = Insulin-Like Factor 1.

Article Snippet: hSSCs were differentiated from hiPSCs as previously described,[ ] with a magnetic activated cell sorting (MACS) step to purify for Thy-1 Cell Surface Antigen (THY1/CD90)-expressing cells. hiPSCs were grown on Matrigel in Minimum Essential Medium Alpha (αMEM, Gibco A10490-01), 1X GlutaMAX (Thermofisher, 35050079), 1X Insulin Transferrin Selenium Liquid Media Supplement (ITS, Millipore Sigma, I13146), 0.2% BSA (Miltenyi Biotec, 130-091-376), 1% penicillin/streptomycin (Sigma Aldrich, P4333), 1 ng/mL human recombinant Fibroblast Growth Factor 2 (FGF2, STEMCELL Technologies, 78003.1), 20 ng/mL animal component-free human recombinant Glial Cell Derived Neurotrophic Factor (GDNF, STEMCELL Technologies, 78139), 0.2% Chemically Defined Lipid Concentrate (Thermofisher, 11905031), and 200 μg/mL L-ascorbic acid (Sigma, A4544).

Techniques: Microscopy, Derivative Assay