Method for in vitro generation of human spermatids

A method of generating human spermatozoa from differentiating spermatogonia (dSPGs) or spermatogonial stem cells (SSCs) in vitro, the method comprising:

• a. culturing human dSPGs or SSCs in a first culture medium of a first culture system at a temperature ranging from about 31° C. to about 33° C. for a first culture period sufficient for entry of the human dSPGs or SSCs into meiosis, wherein the first culture medium comprises:
• i. a nutrient-rich basal medium and a serum or serum substitute, wherein the nutrient-rich basal medium is present at a sufficiently low concentration to provide a nutrient-restricted medium, wherein the sufficiently low concentration of nutrient-rich basal medium ranges from about 8% to about 12% of the total medium volume and the balance comprising a diluent or buffered salt solution at a concentration ranging from about 88% to about 92%;
• ii. retinoic acid at a concentration ranging from about 4 uM to about 6 uM;
• iii. BMP2, BMP4 and BMP7 each at a concentration ranging from about 15 ng/mL to about 25 ng/mL;
• iv. activin A at a concentration ranging from about 90 ng/mL to about 110 ng/mL;
• v. insulin at a concentration ranging from about 40 ug/mL to about 60 ug/mL;
• and
• vi. IWR-endo-1 at a concentration ranging from about 2 uM to about 4 uM;
• and
• b. transferring the cells resulting from culturing in the first culture medium to a second culture medium of a second culture system and culturing the cells using the second culture system at a temperature ranging from about 31° C. to about 33° C. for a second culture period sufficient for the progression of the human spermatocytes arrested before the pachytene stage of meiotic prophase I through completion of meiosis and formation of round spermatids, the second culture system comprising:
• i. a second culture medium comprising
• 1) a nutrient-rich basal medium and a serum or serum substitute, wherein the nutrient-rich basal medium is present at a sufficiently high concentration to provide a nutrient-rich medium, wherein the sufficiently high concentration of nutrient-rich basal medium ranges from about 85% to about 95% of the total medium volume;
• 2) FSH at a concentration ranging from about 15 ng/mL to about 25 ng/mL;
• 3) testosterone at a concentration ranging from about 9 uM to about 11 uM;
• 4) bovine pituitary extract (BPE) at a concentration ranging from about 40 ug/mL to about 60 ug/mL;
• 5) L-Glutamine or a L-alanyl-L-glutamine dipeptide at a concentration ranging from about 0.5 mM to about 5 mM;
• and
• 6) optionally c-KIT receptor ligand (KITLG) at a concentration ranging from about 0.5 ng/mL to about 1.5 ng/mL;
• and
• ii. MEF feeder cells;
• and
• c. transferring the cells resulting from culturing in the second culture system to a third culture medium of a third culture system and culturing the cells in the third culture system at a temperature ranging from about 31° C. to about 33° C. for a third culture period sufficient for generation of human spermatozoa from the round spermatids, wherein the third culture medium comprises:
• i. a nutrient-rich basal medium and a serum or serum substitute, wherein the nutrient-rich basal medium is present at a sufficiently high concentration to provide a nutrient-rich medium, wherein the sufficiently high concentration of nutrient-rich basal medium ranges from about 85% to about 95% of the total medium volume;
• ii. 2-mercaptoethanol at a concentration ranging from about 45 uM to about 55 uM;
• iii. testosterone at a concentration ranging from about 3 uM to about 7 uM;
• iv. retinoic acid at a concentration ranging from about 4 uM to about 6 uM;
• and
• v. conditioned media comprising MEF-conditioned third culture medium, lactate at a concentration ranging from about 0.8 mM to about 1.2 mM, or a combination thereof;
• wherein entry of the human dSPGs and SSCs into meiosis is evidenced by EdU incorporation indicating DNA replication and the appearance of spermatocytes arrested before the pachytene stage of meiosis I as characterized by the presence, absence, or localization of HORMAD1, γH2AX, DMC1, RAD51, STRA8, or any combination thereof;
• wherein progression of the human spermatocytes arrested before the pachytene stage of meiotic prophase I through completion of meiosis and formation of round spermatids is evidenced b by the appearance of cells expressing ACRV1;
• and wherein generation of human spermatozoa from the round spermatids is evidenced by the appearance of flagellar cells.