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Blood, Vol. 109, Issue 3, 1298-1306, February 1, 2007
Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment
Blood Anjos-Afonso and Bonnet
109: 1298
Supplemental materials for: Anjos-Afonso and Bonnet, Vol 109, Issue 3, 1298-1306
Files in this Data Supplement:
- Document S1. Supplemental Materials and methods (PDF, 364 KB)
- Figure S1. Representative pictures from several different experiments (n = 3-6) (PDF, 1.62 MB) -
Unstimulated MStrCs were viewed using light microscopy (a). After 14 days of adipogenic induction, cells were fixed and stained with Oil Red O (b, c; panel b was viewed under black-phase contrast). Osteogenic potential was determined by alkaline-phosphatase staining (d,e). An area of osteoblast-like cluster formation is shown (e). Staining for specific antigens for myogenic (f-h), astrocyte/neuronal (i-l), hepatocyte (m-r), and endothelial (s-t) lineages was performed by immunofluorescence. Myogenic differentiation was confirmed with single staining for dystrophin (TRITC; g) and desmin (Cy3; h). Specific staining for astrocyte/neuronal antigens was performed with NeuN-FITC (j), GFAP-Cy3 (k), and Tau-TRITC (l) antibodies. Hepatocyte and endothelial-like cells were confirmed with staining for CK18 (FITC; n, r), albumin (FITC; o,q), HNF (TRITC; p-r), and VWF (FITC; t) respectively. Cells were counterstained either with hematoxylin (a-e) or DAPI (f-k, m-o, s). Respective isotype controls conjugated with FITC and Cy3/TRITC were also performed for each individual lineage (f,i,m,s). Original magnifications: 10×/0.75 NA dry objective (a-b), 20×/0.75 NA dry objective (d, f-m, o-q, s-t), and 40×/0.75 NA dry objective (c,e,n,r).
- Figure S2. Several examples of cryoembedded bone sections of 4-week-old NOD/SCID mice (PDF, 1.65 MB) -
Bone sections were stained either with mouse IgM immunoglobulins (isotype control; a-b) or with SSEA-1 antibody (d-k). Serial sections show specific staining for SSEA-1 (green cells indicated by the arrows) in the bone marrow (b,d). SSEA-1+ cells can be found throughout the marrow, but they are slightly enriched below the endochondreal-bone area (highlighted area of panel a) in a developing bone. Representative bone sections with marrow stained positive for CD45/CD11b/Ter119/CD31 (c, f k). SSEA-1+ cells are located close to CD45/CD11b/Ter119/CD31+ cells, but they did not stain positively for hematopoietic or endothelial antigens. Immunostainings were performed using appropriate Alexa 488– and Alexa 594–conjugated secondary antibodies. Nuclear counterstaining with DAPI was used. Original magnifications 10×/0.75 NA dry objective (a,c), 20×/0.75 NA dry objective (b,d,g-h,j,k), and 40×/0.75 NA dry objective (e-f,i).
- Figure S3. MAPC culture conditions support SSEA-1+ cells expansion (PDF, 322 KB) -
(A) Both SSEA-1+ and SSEA-1– cells were purified and placed in MAPC culture condition and allowed to expand for 16 doublings (16d) before they were further analyzed for the expression of different surface antigens (n = 3). Both positive and negative cells were capable of expanding under this condition, but only the positive fraction maintained the expression of SSEA-1, KDR, and Trk. We observed a slight overall down-regulation of these antigens after 5 cell doublings (data not shown), but their expression levels were then maintained in further cell doublings. (B) MAPC medium was able to reinduce the expression of Oct-3/4, Nanog, and Rex-1 in SSEA-1+ cells to levels comparable to those found in cells freshly isolated from bone marrow (Figure 1). The SSEA-1– fraction, on the other hand, was negative for these embryonic antigens before and after culture in MAPC culture condition. The expression of Oct-3/4, Nanog, and Rex-1 in SSEA-1+ (S+) and SSEA-1– (S–) cells isolated from mesenchymal cultures was determined by QRT-PCR. The relative mRNA expressions were obtained using ES cells as reference. All quantitative results shown are the mean of fold change (×) from 3 independent experiments. This same condition did not alter the capacity of either SSEA-1+ cells (data not shown; better exemplified with data from the clonally derived populations shown in Figure S5) or SSEA-1– cells to differentiate in vitro (C). FACS analysis of MAPC-expanded SSEA-1– cells (for ~20d) that were induced to differentiate into astrocyte-like (GFAP), hepatocyte-like (albumin and HNF), and endothelial-like (extracellular CD31 and VWF) cells in vitro. The numbers indicate the ratio of MFI values from the specific staining over those from the isotype controls.
- Figure S4. Derivation of clonal SSEA-1+ populations (PDF, 346 KB) -
(A) Single SSEA-1+ cells were sorted from passage 1–adherent cultures, which are mainly composed of CD45/CD11b+ cells. (B) Graphic representation of growth kinetics of 3 surviving clones. (C) Expression of SSEA-1, KDR, Trk, MHC-I, and CD44 on different clonally derived populations and the maintenance of the phenotype of clone-3 cells throughout time.
- Figure S5. In vitro differentiation capacity of clonally derived SSEA-1+ populations (PDF, 301 KB) -
The 3 clonally derived SSEA-1+ populations have different in vitro differentiation capacities. FACS histograms show levels of expression of specific antigens for osteogenic (osteocalcin and BSP II), endothelial (extracellular CD31 and extracellular Tie-2), myogenic (desmin and dystrophin), hepatocyte (albumin and HNF-1), and astrocyte (GFAP) lineages. The expression of these specific antigens was not detected before in vitro stimulation. For all FACS plots shown, gray and white areas are the isotype control and specific antibody staining, respectively. The numbers indicate the ratio of MFI values from the specific staining over those from the isotype controls.
- Figure S6. Differentiation of clone-3 SSEA-1+ cells into different functional cell types of the 3 germline layers in vitro (PDF, 1.24 MB) -
Representative data from 2 to 3 independent experiments that were carried out with cells from 22 to 35 doublings. Cells were used for (a) immunostaining, (b) FACS, and (c) RT-PCR analysis to verify the presence of specific markers for each specific lineage. Clonally derived osteoblasts/osteocytes acquired high levels of alkaline-phosphatase activity and deposited high levels of calcium (d; Dif). These functional features were not observed before induction (d; Undif). Hepatocyte-like cells were able to deposit glycogen (d; PAS assay) and showed cytochrome P450 activity (e; PROD assay). The cytochrome P450 activity in differentiated cells (Dif) can be dramatically induced in the presence of phenobarbital. Endothelial-like cells can form tubular structures in the angiogenesis assay (d) and highly uptake LDL (red staining). Differentiated cells also acquired expression of Tie-2, which colocalized with LDL staining (e). Moreover, they were able to up-regulate different adhesion molecules upon exposure with IL-1 for 6 hours (f; filled gray histograms are the isotype controls, open gray and blue histograms are the specific antibody staining on unstimulated and stimulated cells, respectively). Astrocyte-like cells were able to uptake 3H-glutamate in a Na+-dependent manner (d). Also, differentiated cells acquired glutamine synthetase activity (e) and able to respond to different neurotransmitters such as K+, glutamate, and histamine. These responses were measured by intracellular calcium fluxes using flow cytometry (f). Flow plots show the time course of Ca2+ shifts (200 s) with the basal levels obtained before the addition of any substance (time of addition is indicated by the arrows). Ionomycin was used as a positive control. All quantitative results shown are mean ± SD from 2 independent experiments, each one performed in triplicate. Immunostainings were performed using appropriate Alexa 488– and Alexa 594–conjugated secondary antibodies. Cells were counterstained either with DAPI or hematoxylin. Original magnifications: 10×0.75 NA dry objective (Cd and Ce; Undif Ac-LDL uptake) and others with 20×0.75 NA dry objective.
- Figure S7. Examples of frozen liver sections of engrafted newborn mice stained with MHC-I (Alexa 594; red) and VWF (Alexa 488; green) showing presence of donor-derived endothelial cells (PDF, 1.62 MB) -
Sections were counterstained with DAPI. Original magnifications: 20×0.75 NA dry objective (a-d) and 40×0.75 NA dry objective (highlighted area of panel b). M# denotes the mouse number.
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