Tofacitinib citrate

THY‐1⁺ germ cell cultures were established as described previously.⁸ Briefly, male C57 LacZ pups (5‐8 days post‐partum) were sacrificed and testes were digested using Trypsin‐EDTA (Gibco, USA). Cells were magnetically sorted using CD90.2 beads (Miltenyi, USA) and plated onto Sandos inbred mouse (SIM)–derived 6‐thioguanine‐resistant and ouabain‐resistant (STO) feeders (SNL76/7, ATCC). Germ cell cultures were maintained in a humidified atmosphere at 37°C contains 5% CO₂ with Mouse serum‐free media (mSFM) with 20 ng/mL recombinant human GDNF (R&D, USA), 150 ng/mL recombinant rat GFRα1 (R&D) and 1 ng/mL recombinant human FGF2 (Corning, USA). Other reagents were used as indicated: recombinant human FGF9 (R&D), SB203580, GW788388 and Tofacitinib citrate (all from MedChemExpress, USA). Germ cell counts were determined with a hemocytometer. Feeder cells were identified and discounted from counts based on morphology.²² SSC number was calculated using the following formula, adapted from reference ⁴: $SSCgrowth=\frac{\mathit{Cellsharvested}}{\mathit{Cellsseeded}}\times \frac{\mathit{Colonies}}{{10}^{5}cellstransplanted}$
Here, ‘cells seeded’ indicates the number of cultured THY‐1⁺ germ cells seeded at the beginning of each time period and ‘cells harvested’ is the cell count at the end. ‘Colonies’ indicates the number of distinct colonies counted per testis and is divided by the number of cultured THY‐1⁺ germ cells transplanted per testis.

Glycidol (96%) and ε-caprolactone (99%) were purchased from Acros Organics, (Morris Plains, NJ, USA) and were distilled prior to their use. Tin (II)-2-ethylhexanoate (Sn(Oct)₂) and sulfur trioxide pyridine complex were purchased from Sigma-Aldrich (Hamburg, Germany). Tofacitinib citrate was purchased from Medchemexpress (Monmouth Junction, NJ, USA). Sunitinib was purchased from Selleckchem (Houston, TX, USA). A PD-10 Sephadex^TM G-25 Desalting Column was purchased from GE Healthcare (Stockholm, Sweden). For the purification of the polymer before sulfation, tangential flow filtration (TFF) was used with a 30 kDa molecular weight cut-off (MWCO) regenerated cellulose cassette (Merck, Darmstadt, Germany) in a cassette holder (Sartorius, Göttingen, Germany). A peristaltic pump (Gibson, Nashville, TN, USA) was used to circulate the solution into the system. Benzoylated cellulose dialysis membrane (Sigma-Aldrich, Hamburg, Germany, MWCO = 2 kDa) was used to purify the sulfated polymer.