The defining characteristic of stem cells is their ability for asymmetric division to provide progenitors for specific tissue generation, while maintaining renewal of the stem cell population. LIF protein has been shown to be critical for maintaining embryonic stem cells in an undifferentiated state. UCB HSC in vitro expansion using cytokines has been pursued to augment hematopoietic recovery following UCB transplantation. Our previous studies have shown that a feeder layer of huMSC inhibits UCB HSC proliferation and differentiation during short-term cytokine-driven expansion. We sought to determine whether LIF is secreted by huMSC and if so, at what concentrations, and to elicit its role, if any, in inhibiting cytokine-driven UCB HSC proliferation.
Third passage huMSC were cultured at density 2×10e6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72h and analyzed for LIF secretion by ELISA. UCB was obtained, and MNC were separated on a Histopaque-1077 density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi Biotec) and surface stained for LIF receptor (LIF-R) using anti hLIF-Rα antibody (R&D Systems). LIF-R expression by isolated UCB CD133+ HSC was confirmed by Western blot (n=3). Isolated UCB CD133+ HSCs were plated in 24 well plates at density 3.3×10e3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B. UCB CD133+ HSCs were culture-expanded for 96h with or without recombinant human LIF (10ng/ml) in a combination of cytokines including: IL-3, IL-6, Flt-3L, SCF, G-CSF, and EPO. At 0, 48, and 96h cell counts were obtained. Given variability of LIF & LIF-R expression by primary human CD133 HSC, we compared the expression of key factors in the LIF signaling pathway between the TF-1 cell line (CD34+/CD38+) and the tumorigenic sub-clone, TF-1a cell line which displays a more primitive HSC phenotype (CD34+/CD38−), using quantitative PCR (qPCR). TF-1 and TF-1a cells (ATCC) were propagated as defined by ATCC. TF-1 cultures were supplemented with human GM-CSF (required for growth of these factor-dependent cells). Cell cultures were maintained at density range 0.3–5×10e5/ml for TF-1 cells and 0.3–3×10e6/ml for TF-1a per ATCC guidelines. Culture aliquots containing equivalent numbers of suspended cells from TF-1 and TF-1a cultures were collected by centrifugation and total RNA prepared (Trizol). RNA was quantified by spectrophotometry and equivalent amounts were used to prepare cDNA by reverse transcription. qPCR was performed in 96-well plates using commercially available Taq-Man primers, with GAPDH used as endogenous control. The assays were run on an ABI 7500 Fast system and SDS software. Comparisons to the control transcript were performed using the 2-(ddCt) method, after verifying baseline, threshold, and completion of reactions as indicated by the plateau phase of the amplification curve.
huMSC secreted LIF at all 4 time points, with peak secretion at 48h (mean 52.1(±3.3) pg/ml) (n=3). Surface expression of LIF-R on gated CD133+ cells was 2.61%. At 48h in vitro expansion, higher UCB CD133+ cell counts in cultures without LIF were noted [6.3×10e4 (±0.9)/ml], versus cultures with LIF [4.4×10e4 (±0.8)/ml] suggesting LIF inhibits UCB HSC proliferation. We observed little difference in LIF transcript secreted by TF-1 and TF-1a cells, but a significant down-regulation of LIF-R in TF-1a (RQ=-6.37) compared to TF-1 cells. Similarly, there was a reduction of SOCS3 transcript in TF-1a cells (RQ=-5.23) compared to TF-1 cells. However, expression of Myc, a primary downstream target of LIF-JAK/STAT signaling, did not differ between TF-1 and TF-1a.
LIF secretion by huMSC peaks at 48h at a concentration 3 logs lower than that previously used to inhibit embryonic stem cell differentiation (10ng/ml). LIF exerted inhibitory effects on UCB HSC proliferation at early time points (48h) in cytokine-driven in vitro expansion studies. We observed a down-regulation of the LIF-R and the SOCS3 transcript as might be expected. These data are the first to demonstrate and characterize LIF secretion by huMSC. Further studies are ongoing to further clarify cellular pathways involved in the regulation of LIF signaling on UCB CD133+ HSC differentiation during short-term in vitro cytokine expansion.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.