Leukemia Inhibitory Factor (LIF): Marrow-Derived Human Mesenchymal Stem Cell (huMSC) Secretion and Potential Impact on Umbilical Cord Blood (UCB) CD133+ Hematopoietic Stem Cells (HSC) Differentiation.
Authors: Chapman G. , Banks J.J. , Fanning L.R. , Kozik M. , Finney M.R. , Weitzel R.P. , Greco N.J. , Sakthivel R. , Laughlin Mary J. 
Published: April 2020

Abstract
In vitro expansion of UCB using cytokines has been pursued to overcome the limited stem cell content of a single UCB graft. We have previously demonstrated that a feeder layer of huMSC inhibits UCB HSC differentiation during short-term cytokine-driven expansion in vitro. The protein LIF has been shown to inhibit the differentiation of neurosphere stem cells during 3 week culture over a monolayer of murine stromal cells (C. Shih, et al., 2001). We sought to investigate the hypothesis that LIF secreted by bone marrow (BM) derived huMSCs is involved in inhibition of UCB HSC differentiation during short-term cytokine-driven expansion in vitro. BM derived huMSCs (third passage) were cultured at 2x10^6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72 hours and analyzed for LIF secretion levels by ELISA (Quantikine). LIF secreted by huMSC was noted at all four time points, with peak secretion at 48 h (mean 52.1±3.3 pg/ml) (n=3). UCB was obtained according to institutional guidelines after normal full-term deliveries, collected into bags with citrate dextrose (Allegiance), and MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi) and surface stained for LIF receptor (LIF-R). Surface expression of LIF-R on gated CD133+ cells was 2.61%. Total LIF-R expression in isolated CD133+ cells was further confirmed by Western blot (n=3) using anti-LIF-R antibody (Chemicon). Isolated UCB CD133+ were plated in 24 well plates at 3.3x10^3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B (Gibco). UCB CD133+ were culture-expanded for 96h with or without the addition of recombinant human LIF (Chemicon) (10ng/ml) in a combination of cytokines including: IL-3 (20 ng/mL), IL-6 (20 ng/mL), Flt-3L (100 ng/mL) (R&D), SCF (100 ng/mL), G-CSF (20 ng/mL), and EPO (0.1 U/mL) (Amgen Inc.). At 0, 48, and 96 h cell counts were obtained and flow analysis was performed including surface staining for: CD133, CD34, CD38, HLA-DR, CD33, CD71, and CD41B (Becton Dickinson). At 48 hours, higher cell counts in cultures without LIF were noted 6.3x10^4 ± 0.9/ml, compared with cultures with LIF 4.4x10^4 ± 0.8/ml. However, at 96 h cell counts equalized when comparing cultures with or without LIF at 7.510x10^4 ± 0.9/ml, and 7.8x10^4±0.9/ml respectively (n=6). Surface expression of differentiation markers on gated CD133+ cells did not differ when comparing cultures (n=3). In summary, we observed LIF secretion by MSC peaks at 48 h at a concentration 3 logs lower than that previously used to inhibit stem cell differentiation (10 ng/ml). Although LIF-R is expressed on CD133+ HSC, no difference in cell expansion nor surface phenotype of UCB 133+ cells was observed at early time points (96h) during expansion in cytokines with or without the addition of LIF. Taken together, huMSC mediated inhibition of cytokine-driven UCB HSC differentiation is not attributable to LIF secretion alone and may require direct cell contact between UCB CD133 HSC and huMSC. Studies are ongoing to determine whether LIF may augment huMSC-based UCB CD133 expansion.