The Generation of Programmable Cells of Monocytic Origin Involves Partial Repression of Monocyte/Macrophage Markers and Reactivation of Pluripotency Genes

Abstract

We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into hepatocyte-like cells using appropriate differentiation media. Phenotype conversion required prior in vitro culture in the presence of M-CSF, IL-3, and human serum, during which the cells acquired a state of plasticity, so were termed "programmable cells of monocytic origin" (PCMO). Here, we have further characterized the process of PCMO generation with respect to markers of monocyte-to-macrophage transition and pluripotency. During a 6-day culture period, various monocyte/macrophage differentiation markers were down-regulated being indicative of a process of partial dedifferentiation. Dedifferentiation and hepatic redifferentiation also proceeded in highly purified monocyte preparations, albeit with different kinetics, suggesting that the presence of nonmonocytes, or soluble factors derived from them, is not essential in order for monocytes to acquire a multipotent state. PCMOs expressed various markers of human embryonic stem cells with early induction of NANOG and OCT4. Expression of the pluripotency-associated OCT4A isoform was paralleled by a global rise in histone H3 methylation on Lys-4, a marker of active chromatin, and coincided with peak sensitivity to tissue-specific differentiation. These results show that peripheral blood monocytes can be induced in vitro to transiently acquire stem cell-like properties and concomitantly a state of increased differentiation potential toward the hepatocytic phenotype.

EGF and HB-EGF enhance the proliferation of programmable cells of monocytic origin (PCMO) through activation of MEK/ERK signaling and improve differentiation of PCMO-derived hepatocyte-like cells

“…p47 phox an essential subunit of the reactive oxygen producing enzyme NAD(P)H oxidase and upregulate markers of pluripotency, e.g. Nanog [6]. We have examined the effect of EGF and HB-EGF on the expression of p47 phox by immunoblotting (Figure 3A) and on the expression of Nanog by qPCR (Figure 3B).…”

“…Ideally, a modification of the PCMO generation procedure, e.g. by addition of growth-stimulatory factor(s), should not only enhance mitotic activity but also the plasticity of PCMOs in such a way that the resulting NeoHepatocytes become more hepatocyte-like [6]. Interestingly, a subpopulation of human monocytes that proliferates in vitro in response to M-CSF has been suspected to be less mature and hence more stem cell-like than other monocytes [7].…”

Monocytes Do Not Transdifferentiate into Proper Osteoblasts

“…Ungefroren et al confirmed a change of the monocytes phenotype towards a more stem-cell-like appearance during a 6-day dedifferentiation treatment. The cells downregulated some monocyte-defining gens and started to express stem cell markers over time [19]. Recently, our group demonstrated that dedifferentiation of monocytes towards PCMOs with subsequent hepatogenic differentiation can be significantly improved by the use of the patient's autologous serum instead of human AB serum or fetal calf serum (FCS) [3].…”

Generation of Monocyte-Derived Insulin-Producing Cells from Non-Human Primates According to an Optimized Protocol for the Generation of PCMO-Derived Insulin-Producing Cells

“…Monocytes have also been used by our group (12) and by Hur et al (13) to generate insulin-producing cells. Our protocol includes growth factors treatment for subsequently undergoing dedifferentiation followed by programmability (12).…”

“…Our protocol includes growth factors treatment for subsequently undergoing dedifferentiation followed by programmability (12). We have previously shown that these programmable cells of monocytic origin (PCMO) can then differentiate into insulin-producing cells (14) and hepatocyte-like cells (neo-hepatocytes) (15).…”