PMC

Immunohistochemical staining for Oct 3/4 and Nanog in sections from tumor biopsies of chondrosarcoma and osteosarcoma. One representative osteosarcoma (OS 154) and chondrosarcoma (CS 187) and a positive control, human fetal testes, are shown, as indicated. CS 187 shows a single nucleus positive (brown) for Oct 3/4 and multiple nuclei positive (brown) for Nanog in lung metastasis from a chondrosarcoma. OS 154 sections demonstrate scattered Oct 3/4 nuclear staining and near-complete nuclear Nanog staining in a primary fibular osteosarcoma. Twenty-six-week fetal testes with scattered Oct 3/4 and Nanog nuclear staining are shown as positive controls.

Phase contrast images of monoclonal sarcospheres formed from self-renewing cells from bone sarcoma. Monolayer cultures of cells isolated from bone sarcoma were seeded at clonogenic density into N2 medium with 1% methylcellulose (see section) and cultured for 10 to 14 days. Under these conditions, spheres form at a frequency of 1/100 to 1/1000 cells. (A) Representative image of a sarcosphere in the suspension culture. Cells within the sphere show a compact undifferentiated morphology. (B) Sarcosphere removed from the suspension culture and allowed to attach to a substratum. Adherent cells can be seen expanding from the sphere.

Analyses of bone sarcoma cultures for the expression of genes of endodermal and ectodermal lineages. (A) RT-PCR analyses of adherent and sarcosphere cultures as described in for the transcription of endoderm-associated genes (Gata-4, Gata-6, and AFP) and the neuroectoderm marker β-III tubulin. Primers for β-actin were used as positive reaction controls, as indicated. The control lane represents parallel RT-PCR reactions performed without reverse transcriptase. (B) Western blot analyses for the expression of β-III tubulin and AFP from lysates of adherent cultures are shown in panel A, demonstrating protein expressions of endoderm and neuroectoderm-associated genes. (C and D) Expression of β-III tubulin in tissue specimens from bone sarcomas as detected by immunohistochemistry, and in adherent cultures (E and F) as demonstrated by immunocytochemistry. In panels C and D, arrows indicate regions of positive staining. In panels E and F, areas of β-III tubulin staining are seen in red. Nuclei were counterstained blue using Hoechst's stain.

Multipotent cells in bone sarcoma and the expression of mesenchymal lineage genes. (A) Immunocytochemical staining for Stro-1, a cell surface marker of mesenchymal stem cells, in cultures of osteosarcoma (OS 99-1) and chondrosarcoma (CS 828) cells. Positive staining is shown in orange. Counterstaining for F-actin is seen in green. Following incubation in osteogenic or adipogenic media to induce differentiation along mesenchymal lineages, the respective cultures were analyzed for mineralization by von Kossa stain or for lipid vacuoles by Oil Red O stain. As shown, both cultures showed focal staining for osteogenic and adipogenic differentiation. (B) Semiquantitative RT-PCR analyses for the expression of mesenchymal lineage genes in adherent and sarcosphere (SP) cultures from osteosarcoma (OS) and chondrosarcoma (CS). Reaction products for the respective cultures using primer pairs specific for RUNX2, RUNX3, ALP, osteocalcin (OS Ca), and bone sialoprotein (IBSP) are shown, as indicated. Reactions using primer pairs for β-actin were used as positive controls and also to normalize band intensities between samples. With the exception of ALP, which was preferentially expressed in the adherent cultures, no significant differences were seen between the different culture types. All genes assayed were expressed at detectable levels in both conditions.

Genes specific to ES cells show increased expression in sphere cultures derived from bone sarcomas. (A) Monolayer and sarcosphere (SP) cultures initiated from five osteosarcoma (OS) and three chondrosarcoma (CS) biopsies were analyzed for the expression of Oct 3/4, Nanog, and STAT3 using semiquantitative RT-PCR. β-Actin expression was used as a positive control. Sphere cultures demonstrate increased transcription of both Oct 3/4 and Nanog over adherent cultures. STAT3 expression was uniform between both culture types. (B) Relative band intensities for Oct 3/4 and Nanog for each culture from (A) were quantitated by densitometry, normalized relative to β-actin, and plotted on the graph (Oct 3/4, x-axis; Nanog, y-axis). As indicated by the grouping, the sphere cultures of each sarcoma showed a significantly greater expression of both Oct 3/4 and Nanog than adherent cultures (P < .05, Pearson's correlation coefficient). (C) Western blot analysis of lysates from representative bone sarcoma cell cultures for the protein expression of Oct 3/4, STAT3, and activated (phosphorylated, p) STAT3. β-Actin was used as a positive control for loading, membrane transfer, and immunoblotting. All cultures showed positive staining of protein bands of appropriate sizes, as indicated. (D) Small (left) and large (right) sarcospheres were embedded in paraffin and stained using immunohistochemistry for Oct 3/4 and Nanog, as indicated. Small spheres show an intense staining of cells in the periphery (arrows). Large spheres show similar numbers of darkly staining cells (arrows), with dramatically increased numbers of poorly staining cells in the interior of the sphere.