Stem Cells Dev. 2014 Apr 1;23(7):729-40.

Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

Mineo Iwata1, Richard S. Sandstrom2, Jeffrey J. Delrow1, John A. Stamatoyannopoulos2,3, and Beverly Torok-Storb1

Fred Hutchinson Cancer Research Center , Seattle, Washington.

 

Abstract

Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146(+) and CD146(-) stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146(+) and CD146(-) cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146(+) CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146(-) cells express markers associated with pre-dendritic cells. Importantly, when CD14(+) monocytes are cultured in contact with the CD146(+) cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146(-) cells does not differ from that obtained when monocytes are cultured with CD146(-) CM. These in vitro results show that the CD146(+) marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146(+) cells and CD68(+) cells associated with these upregulated proteins.

PMID: 24131213

 

Supplement:

Studies designed to identify cells contributing to the hematopoietic microenvironment (ME) are seriously limited if they only focus on defining the functions of isolated cell populations. Although such studies have identified a number of cell types implicated in ME function, including endothelial cells, fibroblasts, osteoblasts, and monocyte / macrophages, exactly how the cells function in concert has not been addressed. Since none of these cells act in isolation we hypothesized, they should be combined and studied as ME units. Our initial studies showed ME units composed of fibroblasts and monocytes have transcriptome profiles that cannot be explained by adding their individual transcriptomes. Further complexity comes from the fact that there are at least two populations of fibroblasts that result in functionally different ME units when combined with the same monocytes.

The monocyte- derived macrophage is gaining acceptance as a critical component of the ME. In addition to the well-established monocyte- derived osteoclast and the “nurse cell “of the erythroblastic island, the “blanket-cell” which covers areas of myelopoiesis in long term marrow cultures may also be monocyte derived. Using a transgenic mouse model in which eGFP expression is under the control of the CD68 promoter 2, it is possible to visualize green blanket cells in mouse primary long term cultures (Figure 1). Given that CD68 is specific for tissue macrophages we hypothesize that the blanket cell of the long term culture is a specialized tissue macrophage. In vivo relevance is inferred by visualizing CD68 positive cells with veil-like processes in marrow biopsies in which a normal human marrow biopsy is stained for CD68. (Figure 2)

Given that the monocyte-derived macrophage plays a significant role in the ME, and the monocyte in turn is derived from the hematopoietic stem cell (HSC), it is easy to understand how a defect in the HSC could give rise to a dysfunctional ME. This might explain why some diseases (e.g. aplastic anemia and myelodysplasia) which are in some cases attributed to compromised ME function, can be cured by HSC transplantation even though stromal elements like fibroblasts are not transplanted. Hypothetically, in these cases replacing the defective HSC source of compromised monocytes with normal HSC to produce normal monocytes restores normal ME function.

By way of example, we have reported that monocytes from patients with myelodysplastic syndrome (MDS), specifically the monocytes that come from the MDS clone, fail to respond to stromal signals that induce expression of matrix metalloproteinase-9 (MMP-9)1. In this report patient monocytes were cultured with fibroblast conditioned media. Subsequently the MDS-derived monocytes were identified by in situ hybridization for the clonal chromosome marker and then interrogated for MMP-9 expression by immune histochemistry. The results indicated that the monocytes derived from the MDS clone (in this case identified by monosomy 7) had significantly reduced levels of MMP-9. The results also showed a significant correlation between the proportion of these abnormal monocytes and marrow cellularity in 27 MDS patients. Given the role of MMP-9 in facilitating the egress of cells from marrow, it is reasonable to speculate that the reduced expression of this protease in the majority of monocytes contributed to failed egress resulting in marrow hypercellularity. However as discussed above, monocyte-derived macrophages participate at many levels in the hematopoietic regulatory hierarchy, consequently there are potentially many ways abnormal macrophages can compromise the system.

 

Contact:

Beverly Torok-Storb, PhD

Member

Clinical Research Division

Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., D1-100, P.O. Box 19024, Seattle, Washington 98109-1024, United States

btorokst@fhcrc.org

 

References:

  1. Iwata M, et al. Reduced expression of inducible gelatinase B/matrix metalloproteinase-9 in monocytes from patients with myelodysplastic syndrome: correlation of inducible levels with the percentage of cytogenetically marked cells and with marrow cellularity. Blood 2007;109(1):85-92.
  2. Pillai,M.M., Hayes,B., Torok-Storb,B. Inducible transgenes under the control of the hCD68 promoter identifies mouse macrophages with a distribution that differs from the F4/80 and CSF-1R expressing populations. Hematol. 37: 1387-1392, 2009.

 

BTS Fig1

Figure 1. Inverted confocal microscopy of a viable mouse long term marrow culture in which eGFP expression is under the control of the CD68 promoter. Both the transgenic mouse and photomicrograph provided by Manoj Pillai, MBBS currently at Yale University.

 

 

BTS fig2

Figure 2. Sections of a B5-fixed bone marrow biopsy from a healthy human donor incubated with a monoclonal antibody to CD68. Labeling was detected with EnVision Plus/HRP and NovaRed substrate. Image made available by Manoj Pillai, MBBS currently at Yale University.

 

 

 

 

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