Limits...
Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice.

Nilsson SK, Dooner MS, Weier HU, Frenkel B, Lian JB, Stein GS, Quesenberry PJ - J. Exp. Med. (1999)

Bottom Line: This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections.Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes.Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center and the Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA. s.nilsson@pmci.unimelb.edu.au

ABSTRACT
Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

Show MeSH
FISH of femoral  marrow sections. Panel A, male  control showing positive Y  chromosomes (m; deep red/orange). Panel B, female control  (f  ); autofluorescence appears yellow. Panel C, transplant recipient 6 wk after transplant of 1.2 ×  108 male marrow cells. Sections  are viewed using a dual red and  green filter, and therefore have  no counter-staining. Marrow chimerism was 23.9 ± 2.7% (n = 5),  as determined by Southern blot  analysis (Fig. 2). Panel D, same  as C viewed using a DAPI filter.  Note complete lack of auto fluorescence in positive cell. bm, bone  marrow; d, positive bone cell  of donor origin; e, endosteum;  p, periosteum. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192923&req=5

Figure 1: FISH of femoral marrow sections. Panel A, male control showing positive Y chromosomes (m; deep red/orange). Panel B, female control (f  ); autofluorescence appears yellow. Panel C, transplant recipient 6 wk after transplant of 1.2 × 108 male marrow cells. Sections are viewed using a dual red and green filter, and therefore have no counter-staining. Marrow chimerism was 23.9 ± 2.7% (n = 5), as determined by Southern blot analysis (Fig. 2). Panel D, same as C viewed using a DAPI filter. Note complete lack of auto fluorescence in positive cell. bm, bone marrow; d, positive bone cell of donor origin; e, endosteum; p, periosteum. Bars, 20 μm.

Mentions: In situ detection of individual cells using FISH on sections of paraffin-embedded whole murine femurs was done as previously described (16). In brief, mice were perfused with 4% paraformaldehyde at physiological pressure into the descending aorta. 5-μm sections were permeabilized using proteinase K enzymatic digestion. Sections were hybridized with a digoxigenin-labeled Y chromosome–specific painting probe (17) and stained using antidigoxigenin rhodamine-Fab fragments. Levels of autofluorescence were determined by counter-staining in 0.2 μmol DAPI (4,6-diamidino-2-phenylindole) (see Fig. 1 d). Sections were mounted in Vectashield (Vector Labs.).


Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice.

Nilsson SK, Dooner MS, Weier HU, Frenkel B, Lian JB, Stein GS, Quesenberry PJ - J. Exp. Med. (1999)

FISH of femoral  marrow sections. Panel A, male  control showing positive Y  chromosomes (m; deep red/orange). Panel B, female control  (f  ); autofluorescence appears yellow. Panel C, transplant recipient 6 wk after transplant of 1.2 ×  108 male marrow cells. Sections  are viewed using a dual red and  green filter, and therefore have  no counter-staining. Marrow chimerism was 23.9 ± 2.7% (n = 5),  as determined by Southern blot  analysis (Fig. 2). Panel D, same  as C viewed using a DAPI filter.  Note complete lack of auto fluorescence in positive cell. bm, bone  marrow; d, positive bone cell  of donor origin; e, endosteum;  p, periosteum. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192923&req=5

Figure 1: FISH of femoral marrow sections. Panel A, male control showing positive Y chromosomes (m; deep red/orange). Panel B, female control (f  ); autofluorescence appears yellow. Panel C, transplant recipient 6 wk after transplant of 1.2 × 108 male marrow cells. Sections are viewed using a dual red and green filter, and therefore have no counter-staining. Marrow chimerism was 23.9 ± 2.7% (n = 5), as determined by Southern blot analysis (Fig. 2). Panel D, same as C viewed using a DAPI filter. Note complete lack of auto fluorescence in positive cell. bm, bone marrow; d, positive bone cell of donor origin; e, endosteum; p, periosteum. Bars, 20 μm.
Mentions: In situ detection of individual cells using FISH on sections of paraffin-embedded whole murine femurs was done as previously described (16). In brief, mice were perfused with 4% paraformaldehyde at physiological pressure into the descending aorta. 5-μm sections were permeabilized using proteinase K enzymatic digestion. Sections were hybridized with a digoxigenin-labeled Y chromosome–specific painting probe (17) and stained using antidigoxigenin rhodamine-Fab fragments. Levels of autofluorescence were determined by counter-staining in 0.2 μmol DAPI (4,6-diamidino-2-phenylindole) (see Fig. 1 d). Sections were mounted in Vectashield (Vector Labs.).

Bottom Line: This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections.Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes.Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center and the Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA. s.nilsson@pmci.unimelb.edu.au

ABSTRACT
Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

Show MeSH