First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02375
Journal of Cell Science 118, 2441-2450 (2005)
Published by The Company of Biologists 2005
Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo
Heather MacPherson1,
Pamela Keir1,
Sheila Webb1,
Kay Samuel2,
Shelagh Boyle1,
Wendy Bickmore1,
Lesley Forrester3 and
Julia Dorin1,*
1 MRC Human Genetics Unit, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK
2 Scottish National Blood Transfusion Service, John Hughes Bennett Laboratory, Edinburgh, EH4 2XU, UK
3 John Hughes Bennett Laboratory, University of Edinburgh, Edinburgh, EH4 2XU, UK
View larger version (46K):
[in a new window]
|
Fig. 1. Isolation and characterisation of SP cells from bone marrow. SP cells were isolated from hoechst 33342 stained ROSA26 bone marrow by flow cytometry sorting. (A) SP cells from bone marrow were identified and sorted as defined by gate R2. (B) Bone marrow-derived SP cells were found to be a defined population of cells that exhibited low side scatter. (C) Analysis of SP cells shows that 69.16% of the cells expressed Sca-1 whereas 4.20% expressed Gr-1. (D) Analysis of hematoxylin stained SP cells showed them to have low cytoplasm to nuclear ratios.
|
|
View larger version (26K):
[in a new window]
|
Fig. 2. High-level of re-engraftment in animals following SP tail vein injection. Bone marrow from the female mice that had undergone lethal irradiation followed by tail vein injection of 104 SP cells isolated from ROSA26 bone marrow was incubated with FDG to assess the level of engraftment of the bone marrow by the male donor cells. The levels of engraftment were assessed 3 months post SP cell infusion and 7 days post tracheal instillation with 2% polidocanol. Levels of engraftment were calculated as the number of cells that were able to convert FDG to its fluorescent product and could be measured by flow cytometry analysis. (A) Both the positive (ROSA26) and negative (CBA/Ca) bone marrow controls were found to contain 72.98% and 3.3% fluorescent cells respectively. Bone marrow from the experimental mice (SP1-SP5) were found to contain similar levels of fluorescent cells (mean 72.68%) to the positive ROSA26 bone marrow control demonstrating high-levels of bone marrow engraftment. (B) Flow cytometry profiles show the shift in fluorescence obtained from the bone marrow of mice SP1 to SP5 compared with the control samples.
|
|
View larger version (85K):
[in a new window]
|
Fig. 3. PCR analysis of embryos derived from SP injected blastocysts. Cells from C57Bl/6 (WT) mice and ROSA26 (R) mice were mixed at different concentrations, DNA extracted and PCRs performed to assess the ability to detect LacZ DNA by PCR. The resulting gel showed that the ability to detect LacZ DNA is lost at greater than 1:1000 dilution with wild-type (WT) DNA (A). DNA was prepared from tissue obtained from embryos (a-g) that resulted from blastocyst injections with ROSA26 bone marrow SP cells and analysed by PCR for the presence of LacZ DNA and WT DNA. DNA was extracted from embryonic tissue that appeared to show blue X-gal positive staining. PCR controls are shown in panel (B). The resulting gels showed no presence of LacZ DNA (C) whereas WT DNA was detected (D). C, water control; M, molecular mass marker; R, ROSA26 DNA; WT, wild-type DNA.
|
|
View larger version (57K):
[in a new window]
|
Fig. 4. SP cells do not contribute to primary epithelial air liquid interface cultures. Primary epithelial cells were isolated from the tracheas of ROSA26 (A) and C57Bl/6 (B) mice and shown to form differentiated mature tracheal epithelium when 2x105 cells were plated onto a transwell insert. The presence of ROSA26 cells was assessed by X-gal staining (A) of the cultures. However, when 2x104 ROSA26 SP cells isolated from bone marrow were mixed with 2x105 unmarked C57Bl/6 tracheal epithelial cells under the same culture conditions, they were not found to contribute to the formation of the tracheal epithelium (C) as assessed by lack of blue cells following staining with X-gal. These experiments were performed four times with three replicates in each experiment. The black dots that appear on the photomicrographs are transwell pores. Magnification x40.
|
|
View larger version (72K):
[in a new window]
|
Fig. 5. SP cells do not contribute to sub-cutaneous tracheal grafts. Analysis of sections of normal mouse trachea (A) by H&E staining show an intact epithelial layer. De-nuded mouse tracheas (B) show no presence of epithelial cells. (C) De-nuded tracheas instilled with 105 primary epithelial cells developed an intact epithelial layer after sub-cutaneous grafting for 9 days. (D) Grafts that were seeded with 105 C57Bl/6 tracheal epithelial cells and 2x104 ROSA26 bone marrow SP cells showed re-epithelialisation of the trachea at 42 days post subcutaneous grafting. However, as shown by X-gal staining of the sub-cutaneous graft, the bone marrow SP cells did not contribute to the re-epithelialisation of the graft after 42 days as no blue ROSA26-derived cells were observed (E). Bar, 50 µM.
|
|
View larger version (67K):
[in a new window]
|
Fig. 6. Tracheal epithelial stripping following polidocanol treatment. Analysis of sections of normal mouse trachea (A) by H and E staining shows an intact epithelial layer. (B) Trachea from mouse 24hr after 10 µl 2% polidocanol treatment showed that the epithelial layer had been removed. Bar, 50 µM.
|
|
View larger version (72K):
[in a new window]
|
Fig. 7. No X-gal positive cells in tracheas of mice transplanted with ROSA26 bone marrow-derived SP Cells. X-gal staining was used to detect the presence of ROSA26 donor-derived cells, which could be visualised as blue cells as shown in ROSA26 tissue (A,B). The presence of blue X-gal stained ROSA26 bone marrow-derived donor cells was not detected in the tracheas of tissues removed from the mice transplanted with the ROSA26 bone marrow-derived SP cells (C,D). (A-D) Bar, 50 µM. LacZ DNA was detected by PCR in DNA extracted from tracheal section of the five ROSA26 SP transplanted mice (SP1-SP5) (E). Positive Y chromosome PCR of the DNA extracted from the tracheal sections confirmed the presence of male donor cells (F). C, water control; M, molecular mass marker; R, ROSA26 DNA; WT, wild-type DNA.
|
|
View larger version (87K):
[in a new window]
|
Fig. 8. Y-chromosome-positive cells in female recipients of male bone marrow-derived SP cells. (A-D) Y Chromosome FISH carried out on sections of the tracheal epithelium showed the presence of male donor cells (TxRd; red) located on the epithelia of the tracheas that were isolated from female mice transplanted with male bone marrow-derived ROSA26 SP cells. Bar, 10 µM.
|
|
View larger version (74K):
[in a new window]
|
Fig. 9. Cytokeratin staining and Y chromosome FISH. The Y chromosome positive donor cells (TxRd; red) that were detected in the tracheas of the mice that had undergone transplantation with bone marrow-derived SP cells were immunostained for cytokeratin expression (FITC; green). Photomicrographs (A,B) show Y chromosome FISH staining of a male trachea followed by immunohistochemistry to detect cytokeratin expression on the trachea which is normally widespread. Image C shows a Y chromosome positive donor cell that is found on the tracheal epithelium that does not express cytokeratin. Photomicrographs (D-F) show the DAPI, FITC and TxRd images of micrograph C highlighting the lack of cytokeratin staining on the donor cell. (G-J) shows a donor cell that is cytokeratin positive (arrowhead) and one that appears to be cytokeratin negative (arrow) with photomicrographs (H-J) showing DAPI, FITC and TxRd images respectively of micrograph G. (K) Confocal image of Y chromosome and pan cytokeratin positive cells (arrowheads). Bars, 50 µM (A,B); 5 µM (C-K).
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2005
