Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells
Figure 2
Transformed HS-5 cells engraft and sustain hematopoiesis in NOD-Scid mice.
(A) Gamma camera imaging studies of NOD-Scid mice. Frontal (a, b) and lateral (c) views 3 days (a) and 3 weeks (b and c) after intravenous injection of 105 untreated or Aza plus GF–treated HS-5 cells, stably transfected with hemagglutinin-A-tagged human somatostatin receptor 2A (SSTR2A) gene. Thirteen MBq (350 µCi) 111indium-octreotide was injected intravenously 24 hr prior to imaging. Radioactive signals were detected only in the bladder and kidneys of mice that had not been injected with HS-5 cells (control; first two columns). In contrast, radioactive signals were detected in the calvaria and spine at 3 days (a) and the limbs and/or spine at 3 weeks (b, c) after injection of Aza plus GF–treated HS-5 cells in 8 out of 8 animals (images of 5 animals are depicted). Limb and spine signals were prominently increased after 3 weeks. Weak signals were detected at 3 weeks in the spines of mice that were injected with untreated HS-5 cell (columns 3 and 4; 2 of 4 animals are depicted). (B) Left panel: HLA-ABC immunofluorescence of bone marrow smears obtained from NOD-Scid mice 3 weeks after intravenous injection of untreated and Aza plus GF-treated HS-5 cells. Eight mice were injected with Aza plus GF-treated HS-5 cells and 8 mice with untreated HS-5 cells. Bone marrow analysis was performed separately on every mouse. Representative data are depicted. Random fields were scanned and a total of 400 cells, either HLA-positive or -negative, were counted. As shown in panel (d), 18.5% of marrow cells of mice injected with Aza plus GF-treated HS-5 cells were HLA-ABC positive. White arrows point to the positive cells. The insert is a magnification of an HLA-ABC-positive cell. The large cell (arrow head) is a micro-megakaryocyte. No positive staining was detected when slides of marrow cells from Aza plus GF-treated HS-5 cells were stained with the isotype antibody (a) or on slides of marrow cells obtained from mice that were injected with untreated cells (b) or mice that received no injection (c). Right panel depicts a field (X 1000) of HLA-ABC-stained bone marrow cells from a mouse that were injected with Aza plus GF-treated HS-5 cells (right) and a field of HLA-ABC-stained bone marrow cells from mouse that was injected with untreated HS-5 cells (left). (C) Flow cytometry analysis of bone marrow cells obtained either from untreated mice, mice that were injected with 105 Aza plus GF-treated HS-5 cells (First generation) or bone marrow cells that were harvested three weeks after injection of 105 first generation bone marrow cells (Second generation). Black line depicts the isotypic control and the percent of antigen-positive cells is depicted in the right upper corners. Similar results were obtained in two different experiments. This analysis was conducted twice with each cohort. Representative results are depicted.