The main finding of the present study is that cells obtained by outgrowth from murine pancreas explants in MesenCult give rise to a population of myelomonocytoid cells endowed with mesenchymal differentiation potential. These cells also stain positive with dithizone, a zinc-chelating agent commonly used to detect insulin-producing cells [46
]. The endocrine differentiation potential of these cells is being addressed in an ongoing study. In the present study, we focus on their phenotype and MSC-like characteristics.
Monocyte-derived cells include macrophages, fibrocytes, dendritic cells, osteoclasts, and adipocytes. Monocytes, unlike macrophages and fibrocytes, do not express CD105 [50
]. Because the isolated population lacks CD105 expression, it appears to have a monocytoid phenotype. This population meets only part of the minimal criteria for defining MSC established by the International Society for Cellular Therapy [53
]: plastic-adherence in standard culture conditions and capacity to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro
(in the present study, multilineage differentiation potential was not demonstrated with single-cell cloning). Regarding the cell-surface marker profile, the minimal criteria for defining MSC, namely, expression of CD105 and CD90 but not CD45, are not met. However, other MSC/stromal markers (CD44 and CD29) and pericyte/perivascular markers (NG2 and CD146) are expressed [42
]. In this regard, it has been shown that human MSCs in several organs originate from pericytes/perivascular cells and express NG2 [54
]. For comparison, previous studies have shown that human islet outgrowths are positive for multiple MSC and pericyte markers (CD105+
, and CD146+
) but negative for CD45.
Pancreas-derived cells cultured in the 3D-MethoCult system exhibit a dendritic or oligodendrocytic-like morphology characterized by multiple branched filaments. Microglia have a CD11b+
phenotype and can be distinguished from primary macrophages on the basis of their CD45 expression level [55
]. Differentiation of mouse BM-derived stem cells toward microglia-like cells has been reported [56
The isolated myelomonocytoid population appears to have an advantage in terms of survival or growth compared with other cells present in the cellular outgrowth from pancreatic explants. These cells may die off and be taken over by the myelomonocytoid component that persists after extended periods of time. The underlying mechanism is unclear. Because MesenCult is a commercially available medium that has been optimized for growth of MSC, the emergence of a myelomonocytoid population over time is somewhat surprising. In a previous study [15
], we used this medium to expand mouse cardiac-derived MSC, which displayed a stable phenotype (Lin−
, and CD31−
) for more than 25 passages. This observation indicates that MesenCult can preserve the phenotype of cultured MSC for extended periods of time, at least under certain circumstances.
The mesenchymal differentiation potential of the myelomonocytoid population may appear at odds with the established CD45−
MSC phenotype [53
]. However, Sordi et al. [41
] recently showed that mesenchymal cells emerging from human pancreatic culture did not result from an epithelial to mesenchymal transition but represented the expansion of a pool of resident MSC located in the periacinar, perivascular, and periductal space. Using a GFP+
BM transplant model, they showed that mesenchymal cells emerging from pancreatic endocrine or exocrine tissue culture originated mainly from the CD45+
BM compartment. Athough these cells expressed negligible levels of islet-specific genes, they improved islet function and neovascularization after transplantation with a minimal islet mass in a mouse model. Kaiser et al. [57
] showed that a small population of BM MSC originated from the CD45+
fraction, whereas the majority was obtained from the CD45−
fraction. MSC from either fraction could be differentiated into adipocytes, osteocytes, and chondroblasts. Additional studies confirmed that MSC can express CD45 under certain conditions [58
]. In some cases, CD45 expression was dramatically downregulated during in vitro
]. As mentioned above, our data suggest that MesenCult may preserve CD45 expression in cultured pancreas-derived myelomonocytoid cells, whereas partial downregulation of CD45 expression was observed in a standard culture medium (DMEM-10% FCS).
Pancreas-derived myelomonocytoid cells form spherical clusters, which is considered a feature of stemness [43
]. However, these spheres are nonclonal. Similar findings have been reported for neural stem cells, as colonies formed by these cells can grow clonal or nonclonal [61
The origin of the isolated myelomonocytoid population remains unclear. It might originate from blood monocytes trapped in the intravascular compartment of tissue explants, as recently shown for CD45+
cells from cardiac explant outgrowths [62
]. It should be mentioned, however, that we were not able to isolate myelomonocytoid cells from murine BM-derived cells using the same culture conditions. Alternatively, this population might originate from pancreas-resident monocytes, monocyte-derived cells, or MSC. In this regard, Freisinger et al. [63
] showed that clonally isolated, adipose-derived MSC cultured in appropriate differentiation media gave rise to cells expressing monocyte/macrophage and early hematopoietic markers.
Our findings are in general agreement with recent reports on multipotent monocytes. Zhao et al. [64
] isolated a subset of adult pluripotent stem cells (CD14+
, and CD45+
) from human peripheral blood monocytes. These cells in appearance resembled fibroblasts, expanded in the presence of macrophage colony-stimulating factor (M-CSF), and could be differentiated into mature macrophages and T lymphocytes, as well as into epithelial, endothelial, neuronal, and liver cells in the presence of appropriate growth factors. Kuwana et al. [44
] and Kuwana and Seta [65
] described human blood monocyte-derived multipotent cells (MOMCs; CD14+
, and type-I collagen+
) that exhibited a fibroblast-like morphology and contained progenitors with the capacity to differentiate into bone, cartilage, fat, skeletal muscle, cardiac muscle, neuron, and endothelium [65
]. Romagnani et al. [68
] described circulating clonogenic, multipotent CD14+
cells that proliferated in response to stem cell growth factors. Ungefroren and Fändrich [69
] reported that the programmable cell of monocytic origin (PCMO) is a potential adult stem/progenitor cell source for the generation of islet cells. Hur et al. [48
] recently showed that human peripheral blood monocytes could be differentiated into insulin-producing cells using the hematosphere culture technique. Collectively, these data suggest that blood monocytes and monocyte-derived cells, although not considered classic adult stem cells, may represent versatile progenitor cells capable of generating multiple types of cells.
Owing to their mesenchymal differentiation potential, pancreas outgrowth-derived myelomonocytoid cells are of potential interest to cell therapy applications even though this aspect was not directly addressed by the present study. Sordi et al. [41
] reported beneficial effects of pancreatic MSC in diabetic mice, as mentioned above. When cotransplanted with a minimal islet mass, these cells improved neovascularization and islet function. This effect was not due to MSC differentiation into insulin-secreting cells, but to MSC-mediated protective effects on transplanted islets. Moreover, Johansson et al. [70
] showed that MSC within composite endothelial cell-MSC-pancreatic islets improved endothelial cell proliferation and sprouting in vitro
. It therefore could be speculated that pancreas-derived myelomonocytoid cells endowed with MSC potential might exert trophic effects on pancreatic islets via paracrine mechanisms, as reported for pancreatic MSC by Sordi et al. [41
]. Further studies are warranted to test this hypothesis and to define the origin and the endocrine potential of the pancreas-derived myelomonocytoid population.