Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-β

Abstract

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

Fig 1. PCMO resume proliferation during a 4-day culture period.

Peripheral blood monocytes cultured in 6-well plates were cultured for up to 4 days (d) under adherent (a) or suspended (s) growth conditions and subjected on the indicated days to staining for Ki67 and immunoblotting of p21^WAF1. Bar, 50 μm. (A) In situ staining of PCMO cultures with Ki67 (red) and DAPI (blue). (B) Quantification of Ki67/DAPI-double positive cells in adhesion and suspension cultures from four different donors. Standard deviations were below 15%. *, p<0.05. (C) kinetics of p21^WAF1 expression. The bands for p21^WAF1 and α-tubulin, used as a loading control, were densitometrically analyzed. Displayed are the normalized values for p21^WAF1 relative to those on day 1 set arbitrarily at 1.0, from one representative donor out of four different donors analyzed in total.

Fig 2. Activin A and TGF-β1 synthesis and secretion during conversion of monocytes to PCMO.

(A) Standard endpoint RT-PCR-based detection for the β_A-subunit of activin and TGF-β1 in PCMO. MW marker, molecular weight marker = 100 bp ladder. (B) Activin A and TGF-β1 levels in culture supernatants of monocytes/PCMO growing adherently (a) or in supsension (s) at different time points during culture. Supernatants, conditioned for 24 h, were taken every day until day 4 and subjected to ELISA specific for activin A or total TGF-β1, and bioassay for TGF-β. Data were calculated after subtraction of background activin A and TGF-β levels (contained in the AB serum) and represent means ± SD from five different donors normalized to 10,000 cells. Asterisks indicate a significant difference relative to the respective levels on day 1. Numbers below the graph indicate the percentage of active TGF-β in the respective samples as determined by bioassay (means ± SD). Differences were significant between day 2 and day 4 in both adherent and suspended cells.

Fig 3. PCMO respond to exogenous activins and TGF-β1 with phosphorylation of Smad2.

(A) PCMO were stimulated on day 4 of culture with 50 ng/ml of either activin A, activin B, or activin AB in the presence or absence of the ALK4/5/7 inhibitor SB431542 (1 μM), the p38 MAPK inhibitor SB203580 (10 μM), vehicle (dimethylsulfoxide, 0.1%), or medium alone (w/o) for 1 h followed by immunoblotting for phosphorylated Smad2C (p-Smad2), total Smad2 (Smad2), and β-actin as a loading control. (B) Responsiveness of PCMO from two different donors to TGF-β1 stimulation. PCMO were stimulated on day 4 with 5 ng/ml TGF-β1 in the presence or absence of SB431542 (1 μM), or vehicle for 1 h followed by immunoblotting for p-Smad2C and Smad2. Data in A and B are representative of four different donors.

Fig 4. Kinetics of Smad activation during PCMO culture.

The activation state of Smad2 (left) and Smad3 (right) in adherent (a) and suspended (s) monocytes/PCMO was assessed at various time points by immunoblotting and ELISA (graphs below blots) for p-Smad2(Ser465/467) and p-Smad3(Ser423/425). Alpha-tubulin was used for normalization of p-Smad2 and p-Smad3 signal intensities from blots and for the respective ELISA data (means ± SD). The p-Smad3 blots had to be exposed for a much longer time than the p-Smad2 blots due to the low expression of Smad3. Numbers below the blots indicate the densitometric values for phospho-Smads normalized to those for α-tubulin. Data are representative of four different donors.

Fig 5. Identification of the autocrine ligands that regulate pluripotency gene expression and proliferation in PCMO.

Effect of SB431542 (SB), follistatin (FS), and anti-TGF-β antibody (α-TGF-β) on day 4 of culture on (A) Smad2C phosphorylation as determined by immunoblotting and ELISA (graph below blot). Co, isotype control. Numbers in brackets indicate the concentrations used (μM for SB and μg/ml for FS and α-TGF-β. (B) Oct4A and Nanog expression as determined by qPCR (graphs) and immunoblotting. Oct4 and Nanog were immunodetected in nuclear proteins (25 μg/lane) from the same day 3 (Oct4) and day 4 (Nanog) PCMO used for qPCR analysis. Successful enrichment of nuclear proteins and equal protein loading was verified with an antibody to histone H4. (C) Effect of FS, and α-TGF-β on Smad3C phosphorylation and p21^WAF1 expression in day 4 PCMO as determined by immunoblotting and ELISA (graphs below blots). (D) Ki67 expression on day 4 of culture. In (B) and (D), SB, FS, and α-TGF-β were used at concentrations of 1 μM, 0.1 μg/ml, and 5 μg/ml, respectively. ELISA data in A and C, and qPCR data in B and D are means ± SD from triplicate samples and were derived from one donor. Data are representative of four different donors. Asterisks indicate a significant difference between vehicle and SB or FS-treated cells and between Co and α-TGF-β-treated cells.

Fig 6. Expression of cell cycle regulating genes in PCMO following neutralization of endogenous activin(s) and TGF-β(s).

QPCR-based detection of the indicated genes after treatment of PCMO cultures with follistatin or anti-TGF-β antibody. Data represent the-fold stimulation relative to untreated cells (follistatin) or isotype IgG1-treated control cells (anti-TGF-β antibody) on day 4 of culture, mean ± SD. Data (means ± SD from triplicate samples) are from one donor and are representative of four different donors. ANAPC2, anaphase promoting complex subunit 2; CDC2, cell division control protein 2; CDK, cyclin-dependent kinase.

Fig 7. Cartoon to illustrate the effects of activins and TGF-βs, their receptors and target genes, and their inhibitors, on pluripotency and growth of adherently growing PCMO.

Stimulatory (↓) and inhibitory (⊥) interactions are depicted by bold and dashed lines to indicate strong and weak interactions, respectively. Ab, antibody.