American Society of Hematology

Figure 1

$GD2 expression on marrow MSCs. (Ai) Photomicrograph of undifferentiated MSCs showing the characteristic spindle shape and adherent properties of the cells. Original magnification, ×40. (ii) Flow cytometry histograms demonstrating the typical expression pattern of surface antigens (—) isotype and control (- - -), as indicated. (iii-v) Immunocytochemical staining demonstrating the differentiation of MSCs into osteoblasts (Alizarin Red stain), adipocytes (Oil Red O stain), and chondroblasts (Alcian Blue stain). (B) Immunocytochemical staining of culture-expanded MSCs for GD2 (i) and staining without the primary anti-GD2 antibody as a negative control (ii). (iii) Flow cytometry histogram showing GD2 expression (bold line) by MSCs and the isotype control (thin line). (iv) Reverse-transcription PCR for GD2 synthase. Results using primers generating a 230-bp product are shown at the top, primers generating a 99-bp product are in the middle, and β2-microglobulin as a control for the quality and quantity of RNA is at the bottom. No RNA indicates a complete reaction omitting the RNA sample; NB, RNA from neuroblastoma cells (positive control), MSC, culture-expanded after marrow derivation; MNCs, RNA from blood mononuclear cells (negative control); No RT, reaction with MSC RNA, but omitting reverse transcriptase. (Ci) Flow cytometry histogram of bone marrow cells for CD45 expression. The R1 gate indicates CD45− cells. (ii) Analysis of the CD45− cells from the R1 gate for CD105 and CD73 expression. The R2 gate indicates the double-positive cells. (iii) Analysis of the CD45−CD105+ CD73+ cells from the R2 gate for GD2 expression. These cells, MSCs from freshly harvested bone marrow, were never in tissue culture. (D) Flow cytometry histograms of GD2 expression on MSCs after serial passage in tissue culture. The experimental and control curves are as indicated in panel B. (E) Immunocytochemical staining of adipose-derived MSCs (i) and a negative control (ii) in which the primary anti-GD2 antibody was omitted. The specimens were lightly counterstained with hematoxylin. Original magnification, ×4. Immunocytochemical staining of foreskin fibroblasts (iii) and a negative control (iv) as for the adipose-derived MSCs. Original magnification, ×4. (Fi) Flow cytometric histograms showing the lack of GD2 expression on unfractionated bone marrow cells (upper left, BM), and on marrow cells expressing CD45 (leukocytes), CD34 (hematopoietic progenitors), CD33 (myeloid cells), CD3 (T-lymphocytes), or CD19 (B-lymphocytes). anti-GD2 antibody (—), isotype control (- - -). (ii) Anti-GD2 immunohistochemical staining of a bone marrow biopsy specimen (left) and MSCs (right, positive control). Both specimens were counterstained with hematoxylin.$

GD2 expression on marrow MSCs. (Ai) Photomicrograph of undifferentiated MSCs showing the characteristic spindle shape and adherent properties of the cells. Original magnification, ×40. (ii) Flow cytometry histograms demonstrating the typical expression pattern of surface antigens (—) isotype and control (- - -), as indicated. (iii-v) Immunocytochemical staining demonstrating the differentiation of MSCs into osteoblasts (Alizarin Red stain), adipocytes (Oil Red O stain), and chondroblasts (Alcian Blue stain). (B) Immunocytochemical staining of culture-expanded MSCs for GD2 (i) and staining without the primary anti-GD2 antibody as a negative control (ii). (iii) Flow cytometry histogram showing GD2 expression (bold line) by MSCs and the isotype control (thin line). (iv) Reverse-transcription PCR for GD2 synthase. Results using primers generating a 230-bp product are shown at the top, primers generating a 99-bp product are in the middle, and β₂-microglobulin as a control for the quality and quantity of RNA is at the bottom. No RNA indicates a complete reaction omitting the RNA sample; NB, RNA from neuroblastoma cells (positive control), MSC, culture-expanded after marrow derivation; MNCs, RNA from blood mononuclear cells (negative control); No RT, reaction with MSC RNA, but omitting reverse transcriptase. (Ci) Flow cytometry histogram of bone marrow cells for CD45 expression. The R1 gate indicates CD45⁻ cells. (ii) Analysis of the CD45⁻ cells from the R1 gate for CD105 and CD73 expression. The R2 gate indicates the double-positive cells. (iii) Analysis of the CD45⁻CD105⁺ CD73⁺ cells from the R2 gate for GD2 expression. These cells, MSCs from freshly harvested bone marrow, were never in tissue culture. (D) Flow cytometry histograms of GD2 expression on MSCs after serial passage in tissue culture. The experimental and control curves are as indicated in panel B. (E) Immunocytochemical staining of adipose-derived MSCs (i) and a negative control (ii) in which the primary anti-GD2 antibody was omitted. The specimens were lightly counterstained with hematoxylin. Original magnification, ×4. Immunocytochemical staining of foreskin fibroblasts (iii) and a negative control (iv) as for the adipose-derived MSCs. Original magnification, ×4. (Fi) Flow cytometric histograms showing the lack of GD2 expression on unfractionated bone marrow cells (upper left, BM), and on marrow cells expressing CD45 (leukocytes), CD34 (hematopoietic progenitors), CD33 (myeloid cells), CD3 (T-lymphocytes), or CD19 (B-lymphocytes). anti-GD2 antibody (—), isotype control (- - -). (ii) Anti-GD2 immunohistochemical staining of a bone marrow biopsy specimen (left) and MSCs (right, positive control). Both specimens were counterstained with hematoxylin.

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