Basic fibroblast growth factor (bfgf)

The prostate cancer cell lines LnCap, PC3 and DU145 were purchased from the Wuhan University Strain Preservation Center (Wuhan, China) and cultured in Ham's F12 medium or Dulbecco's modified Eagle's medium (Corning, US). These cells were supplemented with exosome-free serum Ham's F12 medium or Dulbecco's modified Eagle's medium supplemented with exosome-free serum (Corning, US), 2 ng/mL basic fibroblast growth factor (Corning, US), 2 mM glutamine (Yongjin Biotech, Guangzhou, China), and 10 units/ml penicillin/streptomycin. Cultures were maintained at 37 °C in an atmosphere containing 5% CO₂.

To assess whether immune cues impact hNSC after SCI in the Rag1 mice, we quantified the effect of innate immune cell CM on hNSC fate in vitro. PMN and MØ were isolated from peritoneal cavities of adult female Rag1 immunodeficient mice, as previously described^{78 (link)}. Briefly, mice were stimulated with 12% sodium caseinate (i.p. injection) and were sacrificed either 12–16 h (to collect PMN) or 5 days (to collect MØ) post-injection (Fig. 1b and Supplemental Fig. 3). PMN and MØ (5,000,000 cells/ml) were cultured in hNSC differentiation media (DM). hNSC DM is X-VIVO 15-based media (Lonza, Basel, Switzerland) supplemented with 10 ng/mL GDNF (PeproTech, Rocky Hill, NJ), 10 ng/mL BDNF (PeproTech, Rocky Hill, NJ), 0.1 ng/mL bFGF, 10 μg/mL Ciprofloxacin (Cellgro), 2 ng/mL Heparin, 63 µg/mL NAC, and 1x N2 and 1x B27 supplements (Thermo Fisher Scientific, Waltham, MA). PMN-CM was collected only once after 24 h in culture. MØ-CM was collected once a day for 3 days, and the culture was replenished with fresh media after each collection. Both conditioned media were diluted (1:1) with fresh DM media and used for the hNSC differentiation experiments (UCI 161 cell line, passage 6).

To assess how immune cues impact hNSC after SCI in the Rag1 mice, we quantified the effect of innate immune cell conditioned media (CM) on hNSC fate in vitro. Polymorphonuclear leukocytes (PMN) and macrophages (MØ) were isolated from peritoneal cavities of adult female Rag1 immunodeficient mice, as previously described [74 (link)]. Briefly, mice were stimulated with 12% sodium caseinate (i.p. injection) and were sacrificed either 12–16 hours (to collect PMN) or 5 days (to collect MØ) post-injection (Fig. 1B and Supplemental Fig. 2). PMN and MØ (5,000,000 cells/ml) were cultured in hNSC differentiation media (DM). hNSC DM is X-VIVO 15 based media (Lonza, Basel, Switzerland) supplemented with 10 ng/mL GDNF (PeproTech, Rocky Hill, NJ), 10 ng/mL BDNF (PeproTech, Rocky Hill, NJ), 0.1 ng/mL bFGF, 10 μg/mL Ciprofloxacin (Cellgro), 2 ng/mL Heparin, 63 μg/mL NAC, and 1x N2 and 1x B27 supplements (Thermo Fisher Scientific, Waltham, MA). PMN CM was collected only once after 24hrs in culture. MØCM was collected once a day for three days, and the culture was replenished with fresh media after each collection. Both conditioned media were diluted (1:1) with fresh DM media and used for the hNSC differentiation experiments (UCI 161 cell line, passage 6).

Reprogramming of aHF and nbHF was carried out with episomal reprogramming vectors pCXLE-hOCT3/4-shp53, pCLXE-hSK, and pCLXE-hUL [18 (link)]. Briefly, 1 × 10⁶ cells (between passages 6 and 10) were transfected with a 2.5 µg mix of each vector using a Neon Transfection System (Invitrogen). The conditions for aHF were 1800 V, 20 ms with one pulse, and for nbHF, they were 1650 V, 10 ms with three pulses. After transfection, cells were cultured for 7 days in reprogramming medium in the presence or absence of 30 µM HYD. The reprogramming medium was formulated with DMEM high glucose supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum (Gibco), 10% KnockOut Serum Replacement (Gibco), 1 mM sodium pyruvate (Corning), 1% non-essential amino acids, 3 µM CHIR99021(Sigma-Aldrich) and 0.5 µM A83-01 (Sigma-Aldrich). At day 8, cells were recovered and seeded on mitotically inactivated mouse embryonic fibroblasts (iMEF). Medium was replaced to KnockOut DMEM supplemented with 20% KnockOut Serum Replacement, 2.5 mM Glutagro (Corning), 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol, and 10 ng/mL of basic Fibroblast growth factor (bFGF) (Corning). Colonies were visualized and counted at 25–30 days, and those with characteristics of human ESC-like colonies [19 , 20 (link)] were picked up for further experiments. Cultures were maintained in the conditions mentioned above.

Patient-derived drug-naïve cells were used to obtain drug-resistant melanoma cell lines [28 (link)]. The study was approved by Ethical Commission of Medical University of Lodz (identification code: RNN/84/09/KE). Each patient signed an informed consent before tissue acquisition. Drug-naïve cell lines were named after Department of Molecular Biology of Cancer (DMBC) with consecutive numbers. They were grown in vitro in stem cell medium (SCM) consisting of Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Lonza, Basel, Switzerland), B-27 supplement (Gibco, Paisley, UK), 10 ng/mL basic fibroblast growth factor (bFGF) (Corning, Corning, NY, USA), 20 ng/mL epidermal growth factor (EGF) (Corning, Corning, NY, USA), insulin (10 µg/mL) (Sigma-Aldrich, St Louis, MO, USA), heparin (1 ng/mL) (Sigma-Aldrich, St Louis, MO, USA), 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 µg/mL fungizone B. To obtain cell lines resistant to vemurafenib (PLX) or trametinib (TRA) (Selleck Chemicals LLC, Houston, TX, USA), melanoma cell lines derived from different tumor specimens were cultured in SCM in the presence of increasing concentrations of respective drug for 4–5 months.

The glioblastoma cell lines U87 and T98G were obtained from the American Type Culture Collection (Manassas, Virginia, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). TPC1115 and TPC0411 were obtained from fresh surgical specimens of human primary GBMs and cultured as either monolayer or tumor spheres^{27 (link)} in DMEM/F12 medium supplemented with N2, B27 (Gibco), 20 ng/ml human fibroblast growth factor-basic (bFGF, Sino Biological, Beijing, China), 20 ng/ml epidermal growth factor-basic (EGF, Sino Biological, Beijing, China). All cells were maintained at 37 °C in a humid incubator with 5% CO₂. For sphere formation, cells were cultured in serum-free DMEM/F12 medium containing 20 μl/ml B27 supplements, 5 μg/ml insulin, 20 ng/ml bFGF, and 10 ng/ml EGF at ultra-low attachment plates (Corning). Cells have been authenticated by examining their karyotypes and morphologies. All cells have been tested for mycoplasma contamination by PCR and were verified to be mycoplasma free.

The enrichment of OCSCs and establishment of single cell-forming OCSCs were performed as described previously [19 (link)]. In brief, ovarian cancer cell lines were cultured in ultra-low attachment plates (Corning®, Corning, NY, USA) in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplied with 1% nonessential amino acids (Thermo Fisher Scientific, Waltham, MA, USA), sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit−Haemek, Israel), 10 μg/mL insulin (Thermo Fisher Scientific, Waltham, MA, USA), basic fibroblast growth factor (bFGF; PeproTech, Rehovot, Israel), and human recombinant epidermal growth factor (EGF; PeproTech, Rehovot, Israel). The cells were cultured in suspension, and starting from 14 days, the cultures were examined every day for sphere formation. Spheres were then dissociated and passaged at least eight times in 2 months to generate spheres, which are henceforth referred to as OCSC (SR1 and SR2) cells. For OCSCs differentiation, we transferred OCSCs from ultra-low attachment plates into standard cell culture dishes (Corning®, Corning, NY, USA) and removed growth factors bFGF and EGF. The cells were harvested and extracted for DNA and RNA at Day 8 (AD1), 21 (AD2), 25 (AD3), and 42 (AD4) after OCSCs adhesion.