Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells

Abstract

Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.

FIG. 1.

Growth, expression analysis, and differentiation of OM-MSCs. MSCs were isolated from human OM and cultured until they reached confluency. Phase-contrast micrographs show cells with fibroblast-stromal morphology (A). Flow cytometry analysis of OM-MSCs shows the expression of CD90, CD73, CD166, CD49b, and CD34. Negative controls were stained with the respective isotype (black histograms) (B). OM-MSCs were collected and cultured in osteogenic, adipogenic, and chondrogenic media. After 14 days, cells were stained with Alizarin, oil red, and Alcian blue. Micrographs show the presence of mineralization in nucleus (Alizarin positive) (D), adipocytes (oil red staining) (E), and blue staining [Alcian blue positive, (F)], indicating the osteogenic, adipogenic, and chondrogenic capacity of OM-MSCs. As control, OM-MSCs were cultured in regular medium (C). OM, olfactory mucosa; MSCs, mesenchymal stromal cells. Color images available online at www.liebertonline.com/scd

FIG. 2.

OM-MSCs promote the proliferation and survival of human BMCs. NACs were collected from BMC/OM-MSC co-cultures and the cell number and viability were determined by trypan blue technique. Results show an increase in the number of NACs at 54 days with respect to 13 days of co-cultures (A). Cell viability was >80% at 54 days of co-culture (B). Microscopic evaluation shows proliferation of rounded cells on OM-MSC monolayer, at 13 and 41 days of BMC/OM-MSC co-cultures (C). Results are representative of at least 3 experiments, all with similar results. BMCs, bone marrow cells; NACs, nonadherent cells.

FIG. 3.

OM-MSCs promote in vitro myelopoiesis from human BMCs. NACs were collected, at day 17, from BMC/OM-MSC co-cultures, and the cells were analyzed for their expression of the CD45 marker by flow cytometry (A). Cytospin from NACs and May-Grünwald/Giemsa staining were performed for morphology analysis showing the presence of polymorphonuclear (PMN), monocytes (mono), basophil (Baso), eosinophil (Eos), lymphocytes (Ly), erythroblast (Erythr), and megakaryocyte (Mk) (B). Results correspond to 1 experiment representative of 3 different assays, all with similar results. Color images available online at www.liebertonline.com/scd

FIG. 4.

OM-MSCs promote in vitro lymphopoiesis from human BMCs. NACs were collected from BMC/OM-MSC co-culture, at day 17, and the cells were analyzed for their morphology and surface expression markers. Microscopic evaluation shows the presence of lymphoid cells (A). Flow cytometry analysis shows the presence of cells expressing T [CD3, (B)], B [CD19, (C)], and NK [CD56/CD16, (D)] cell markers. Results correspond to 1 experiment representative of 3 different assays, all with similar results.

FIG. 5.

Detection of CFU-GM progenitors in BMC/OM-MSC co-cultures. NACs were collected from BMCs/OM-MSCs, after 17 days of co-culture, and colony-forming assays were performed. Microscopic evaluation shows the presence of a CFU-GM colony (A). Single CFU-GM colonies were taken from methylcellulose assays and subjected to cytospin, and May-Grünwald/Giemsa staining showing the presence of myeloid cells (B). Flow cytometry analysis of NACs shows the presence of cells expressing the CD34 marker (C). Results correspond to 1 experiment representative of 2 different assays, all with similar results. CFU-GM, colony-forming unit–granulocyte/macrophage.

FIG. 6.

OM-MSCs constitutively express hematopoietic cytokines. Reverse transcription and amplification were performed as described in the Materials and Methods section using specific primers for SCF, GM-CSF, IL-6, IL-11, and IL-7.

FIG. 7.

OM-MSCs maintain mesenchymal phenotype after long-term co-culture with BMCs. BMC/OM-MSC co-cultures were harvested after 54 days of co-culture, and the cells were analyzed for their expression of the CD90 MSC marker by flow cytometry. Negative controls were stained with the respective isotype (gray profile). Histogram analysis demonstrates that OM-MSCs maintain the CD90 expression on their surface. The results correspond to 1 experiment representative of 2 different assays, all with similar results.