Osteoblasts and bone marrow mesenchymal stromal cells control hematopoietic stem cell migration and proliferation in 3D in vitro model

Abstract

Background: Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow-derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs.

Methodology/principal findings: A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34(+) cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34(+) cells was decreased.

Conclusions/significance: Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Figure 1. Characterization of simple and mixed spheroids.

Simple non-induced spheroids are round cell aggregates (A–B) with elongated cells at the periphery (C, E) that show numerous cytoplasmic projections contacting neighbor cells (C), and delicate filaments (arrow) at the cell surface, indicative of ECM deposition (D). Inner cells have cytoplasmic vacuoles (F), irregular nucleus, well-developed rough endoplasmic reticulum (arrow), and many viable mitochondria (arrow heads) (G). Phase contrast (A), SEM (B–D), and TEM (E–G). Simple non-induced (H), and mixed (I) spheroids with osteo-induced BMSC at the center. Note the limit between the two cell populations (arrowhead). H–E. (J) Confocal microscopy of mixed spheroids confirming that CM-Dil labeled osteo-induced BMSC (in red) were confined to the central region. Nuclei were stained with DAPI (blue). (K) Detection of apoptotic cells (in green) in simple non-induced spheroids by TUNEL method after 5 days in culture. Nuclei were stained with DAPI (blue). (L) Correlation between the diameter of simple non-induced spheroids and the number of plated cells after 4 and 11 days of culture. Data represent mean of 3 spheroids per cell number per day. Error bars are standard error of the mean (± SEM). (M–N) Ki-67 expression in simple non-induced (M, full black line) and mixed (N, full black line) spheroids. Dotted lines are isotype control, and grey line (N) represents a cell line (porcine aortic endothelial cell) used as a positive control. Numbers above scale bars represent the value (in micrometers) of each scale bar.

Figure 2. Extracellular matrix distribution and cytoskeleton organization in simple non-induced and mixed spheroids.

(A) Confocal microscopy of paraffin sections stained with H&E showing complex cellular interactions in the center of simple non-induced spheroids. (B–D) Picrosirius staining of simple non-induced (B) and mixed (C–D) spheroids showing, by optical (B–C) and confocal (D) microscopy, collagen fiber deposition restricted to the inner region of mixed spheroids. (E–J) Expression of ECM protein in mixed spheroids. Immunofluorescence staining (in green) for collagen I (E), laminin (F), collagen IV (G), osteopontin (H), and fibronectin (I–J). Osteo-induced BMSC were labeled with CM-DiI (red). A negative control is shown as an insert in (E). (K) Fibronectin expression in simple non-induced spheroids is shown for comparison. (L–O) Expression of α-SMA (L, N) and actin polymerization (M, O, phalloidin staining) in non-induced BMSC. Note the formation of stress fibers in monolayers (L–M) that are absent in 3D cultures (N–O). Nuclei were stained with DAPI (blue). (P–R) α-SMA expression (green) in mixed spheroids. Osteo-induced BMSC were labeled with CM-DiI (red in P, R). Numbers above scale bars represent the value (in micrometers) of each scale bar.

Figure 3. Gene expression profile of BMSC in 2D or 3D cultures.

(A) A representative RT-PCR analysis is shown for osteo-induced BMSC cultured for 4 days as monolayers (2D ind) or as simple osteo-induced spheroids (3D ind), and non-induced BMSC cultured as monolayers (2D) or simple non-induced spheroids (3D). Blots correspond to the transcriptional factor RUNX2, the Notch ligands DELTA1 and JAG1 (Jagged-1), ANGPT1 (Angiopoietin-1), the inhibitor of Wnt pathway, DKK1, and SPP (Osteopontin). (B–E) Semiquantitative analysis is shown for GAPDH, CXCL12 (C), WNT5a (D), and KITLG (E). (n = 3, ± SEM).

Figure 4. Time-dependent migration of CD34⁺ cells into spheroids.

At defined intervals, cells in the supernatant were collected and spheroids were harvested and trypsinized. (A) The proportion of CD34⁺ cells (R2) was determined by flow cytometry, and calculated as percentages of CD34⁺ cells inside the spheroids in relation to the total plated. (B) Dot plot of control spheroids without hematopoietic cells. (C) To distinguish migration from proliferation of cells inside the spheroids, hematopoietic cells were removed after 24 hours of co-culture, and the spheroids were maintained in culture for up to 48 hours. The number of CD34⁺ events inside the washed spheroids (closed symbols) was compared to the number of CD34⁺ events inside no washed spheroids (open symbols). (D) Time-dependent migration of CB CD34⁺ cells into simple non-induced (full line, dots), simple osteo-induced (dotted line, triangles), and mixed (dotted line, circles) spheroids. (E) The migratory profile of BM (closed squares) and CB (open squares) CD34⁺ cells in mixed spheroids is shown. Data are mean ± SEM.

Figure 5. Migration of CD34⁺ cells in mixed spheroids is dynamic.

CB and BM CD34⁺ cells were co-cultured with simple or mixed spheroids for 24 hours. The supernatant was removed and the spheroids were washed and maintained in culture for more 48 hours. Phase contrast microscopy of CB CD34⁺ cells emigrating from mixed spheroids at 24 hours (A) and 48 hours (B). Number above scale bar represents the value (in micrometers) of both scale bars. Percentage of CB and BM CD34⁺ cells that migrated out from mixed and simple osteo-induced spheroids was determined by flow cytometry (E). Unlabeled cells were co-cultured with mixed spheroids for 24 hours and then the supernatants were removed and the spheroids were washed. CFSE labeled CD34⁺ cells were added to these spheroids and the co-cultures were maintained for an additional 48 hours. Representative FACS analysis showing CD34⁺ cells that were CFSE⁻ or CFSE⁺ in supernatants (C) and spheroids (D) after 48 hours of co-culture. (F) Histogram showing the percentage of CD34⁺ cells that were positive or negative for CFSE in the supernatant or spheroids after 24 (open bar) and 48 hours (grey bar). Data represent average percentages (± SEM) of CFSE⁺ and CFSE⁻ among CD34⁺ cells in one experiment with duplicates. Similar results were obtained in a second independent experiment.

Figure 6. CD34⁺ cells localize at the vicinity of osteo-induced BMSC in mixed spheroids.

(A) Semithin section of simple non-induced spheroids after 48 hours of co-culture with CB CD34⁺ cells, showing numerous hematopoietic cells homogeneously distributed throughout the spheroids, even at the center. Methylene Blue. (B) Ultrastructure of the co-cultures, showing a CD34⁺ cell in contact with stromal cell projections. (C–D) Mixed spheroids co-cultured for 72 hours with CB CD34⁺ cells. Hematopoietic cells (arrows) were aligned at the interface of the two stromal cell layers (C, arrowhead) or at the vicinity of osteoid tissue (* in D). H–E. (E–F) Clusters of CD34⁺ cells (inserts) are seen at the vicinity of the osteo-induced BMSC after 48 hours. Immunohistochemistry. (H–J) Confocal microscopy of mixed spheroids co-cultured for 72 hours with CFSE labeled CB CD34⁺ cells (green, H, J). Osteo-induced BMSC were labeled with CM-DiI (red, I–J) and nuclei, that was not confocalized, were stained with DAPI, (blue, G, J). Note that hematopoietic cells are located in close proximity to osteo-induced CM-Dil⁺ BMSC but actually at the transitional region between the two cell populations. Numbers above scale bars represent the value (in micrometers) of each scale bar. (Bars in inserts  = 30 µm).

Figure 7. Colony formation in Methylcellulose and BrdU incorporation.

The sorted CD34⁺ cells from the supernatant or from inside simple non-induced and mixed spheroids after 2 and 7 days of culture were subjected to methylcellulose assay to determine their potential clonal growth as CFU-GEMM, BFU-e and CFU-GM. (A–C) Representative profile of single-cell suspension isolated from co-cultures before (A–B) and after sorting of CB CD34⁺ cells (C). (D–E) Morphology of day-10 colonies (inverted microscope, x 5); inserts shows cellular morphology of individual colonies (MGG staining, x 40 in D, x 60 in E). (F) Clonogenic myeloid potential in methylcellulose of CD34⁺ freshly isolated from CB or co-cultured for 2 and 7 days with mixed or simple non-induced spheroids (* p = 0.0015). Data is representative of two independent experiments with triplicates. Mean ± SEM. (G) FACS analysis of BrdU expression in CB CD34⁺ cells co-cultured for 48 hours with simple non-induced (dotted line) or mixed spheroids (black line). The grey line represents isotype control. (H) The percentage of CD34⁺ cells that were negative for BrdU after co-culture for 2 or 7 days with simple non-induced (grey bars) and mixed (white bars) spheroids is shown. (** p = 0.037; n = 4 ± SEM).