**2. History**

After an injury occurs, a number of extracellular and intercellular responses are initiated and coordinated in order to restore the tissue integrity and homeostasis. Wound healing is a dynamic, interactive process in which cellular components of the immune system, the blood coagulation cascade and the inflammatory pathways are activated. The cells involved including neutrophils, monocytes, lymphocytes, dendritic cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, differentiation and migration (Singer & Clark 1999; Arabi et al., 2007; Gurtner et al., 2008)

Tissue fibroblasts play a key role not only in normal reparative processes, but also in pathological fibrotic processes. In the past decade it has been established that fibroblasts/myofibroblasts, which participate in repair and fibrosis have their origin not only in the fibroblasts already present in the injured tissues, but also may derive from other sources such as mesenchymal and hematopoietic stem cells (Hinz et al., 2007). The notion of a

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 319

Fibrocytes can be obtained and/or differentiated in vitro from the complete peripheral blood mononuclear cell (PBMC) population as well as from an enriched CD14+ population (Abe et al., 2001, Pilling et al., 2009, García-de-Alba et al., 2010). Accordingly, fibrocytes represent one of the variety of cell types that can differentiate from monocytes, including macrophages, osteoclasts and dendritic cells (Wu & Madri 2010; Seta et al., 2010; Castiello

The fibrocytes obtained from human or mouse blood, either from PBMCs or CD14+ enriched cells, are grown commonly in Dubelcco´s Modified Eagle Medium (DMEM) supplemented with 20% human AB serum (HAB) or fetal calf or bovine serum without the addition of any other growth factors. Some authors have reported the use of RPMI instead of DMEM with good results (Curnow et al., 2010). The resulting fibrocyte population (≥95% pure) is then characterized based on the combined expression of extracellular surface markers including cluster of differentiation (CD) antigens, major histocompatibility complex (MHC)-like molecules, extracellular matrix protein (ECM) markers, and chemokines

Table 1. Human fibrocytes surface and intracellular phenotype. Reviewed in Bucala et al.,

1994, Chesney et al., 1998, Abe et al., 2001, Hartlapp et al., 2001

receptors expression patterns (Metz, 2003) (table 1).

et al., 2011).

monocytic fibroblast precursor was first proposed more than a hundred years ago by James Paget, and probably represents the first observations of cells with the molecular features of circulating fibrocytes (Herzog & Bucala 2010). Afterward in the early 1960´s the hypothesis of the blood borne origin of fibroblast appeared again in the literature; of particular significance are the observations of Petrakis and co-workers who reported the in vivo differentiation of human leukocytes into fibroblasts, histiocytes and adipocytes in subcutaneous diffusion chambers (Petrakis et al., 1961). More recently, it was demonstrated that bone-marrow (BM) contributes to the expansion of the broblast population in multiple organs and tissues, including skin, stomach and esophagus using mouse transplantation models, and in human liver fibrosis (Direkze et al 2003, 2004 and Forbes et al 2004). Regarding the lung, a pioneer work published in 2004 described that the collagen-producing broblasts in experimental pulmonary brosis are derived from BM progenitor cells (Hashimoto et al., 2004). While these studies documented the BM origin of at least part of the tissue broblasts during injury, they did not resolve whether these BM derived broblasts were from hematopoietic stem cells (HSCs) or mesenchymal stem cells. Later, through a model of transplantation of clones of cells derived from a single HSC from transgenic enhanced green uorescent protein (EGFP) mice, it was clearly demonstrated that brocytes are derived from HSCs (Ebihara et al., 2006).

The circulating fibrocyte was first described in 1994 by Bucala, in a model of wound healing response, with the surgical implantation of wound chambers into the subcutaneous tissues of mice. The implantation resulted in a rapid influx of peripheral blood cells such as neutrophils, monocytes, and lymphocyte subpopulations within 24 hr. They noticed that 10% of the cells present in the wound chamber, were spindle shaped cells and expressed collagen I, and CD34, (Bucala et al., 1994). The idea that these cells were of circulating origin arose from the observation that their arrival in to the wound chamber was much faster than would be expected by entry of fibroblasts from the surrounding tissue, since the fibroblasts would have to migrate across the permeable plastic layer, enter the wound chamber, and begin matrix deposition, (Bucala, 2008). Hence, the entrance of large numbers of fibroblastlike cells simultaneously with circulating inflammatory cells suggested that this cell population was from peripheral blood origin and not exclusively by slow migration from adjacent connective tissue (Bucala et al., 1994). This new leukocyte sub-population was termed "brocytes", which combines the greek "kytos" referring to cell, and "bro", which is from the latin denoting ber. This nomenclature may lead to some overlap as the term "brocyte" is also used in histopathologic literature as a synonym for "mature" broblasts, and to name a cell constituent of the inner ear spiral ligament, (Quan et al., 2004).

Now it is known that fibrocytes are a hematopoietic stem cell source of fibroblasts/myofibroblasts that participate in the mechanisms of wound healing and fibrosis in many organs (Schmidt et al., 2003; Mori et al., 2005; Ebihara et al., 2006; Andersson-Sjöland et al., 2008; El-Asrar et al., 2008; Strieter et al., 2009).
