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.
Highlights
The use of adult stem cells has been a reasonable therapeutic option for many diseases
Monocytes/ programmable cell of monocytic origin” (PCMO) growing adherently on tissue culture plastic or those from parallel cultures growing in suspension were stained in situ with the proliferation marker Ki67 (Fig. 1A)
The data suggest that adherent PCMO are more proliferation-active than their counterparts growing in suspension
Summary
The use of adult stem cells has been a reasonable therapeutic option for many diseases. Expression of both genes steadily increased during culture and peaked at days 4–5 [4] This coincided with peak proliferative activity of a monocyte subset as measured by cell counting, thymidine incorporation [3], activation of the proliferation-associated extracellular signal-regulated kinase 1/2 (ERK1/2) [6], and the kinetics of cyclin D1 expression (A.H., unpublished observation). This may suggest the possibility that both responses, Oct and Nanog expression, and mitotic activity, are controlled by the same factors. Other possibilities to achieve this goal are the modification of substrate attachment or attachment-free (suspension) culture, and the removal from or neutralization in the conditioned medium of autocrine factor(s) with growth-inhibitory properties
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