Differentiating Stem Cells Mask Their Origins
http://www.100md.com
《干细胞学杂志》
Department of Tumorimmunology, UMC Nijmegen, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands
Key Words. Stem cell differentiation ? Gene silencing ? Transgene expression ? EGFP marking ? Epigenetic reprogramming
Ruurd Torensma, Ph.D., Department of Tumorimmunology, UMC Nijmegen, Nijmegen Center for Molecular Life Sciences 187, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Telephone: 31-24-3610544; Fax: 31-24-3540339; e-mail: r.torensma@ncmls.kun.nl
Genetic marking of stem cells is a widely used method to trace transplanted cells in a nonmarked recipient. There is considerable debate as to whether organ-specific stem cells can give rise to cells of other organs (transdifferentiation). The number of marked hematopoietic stem cells that can be traced in a specific organ is rather low (less than one in one million cells), raising doubt as to whether the transdifferentiation of stem cells is a likely event or whether it only occurs in a very few instances. Cell fusion of damaged muscle cells with stem cells could also explain the low number of marked cells. Here, we demonstrate that expression of a marker gene, in this case, the gene encoding green fluorescent protein (GFP), is dependent on the differentiation status of the stem cell. T cells residing in the thymus of GFP transgenic mice do not express this transgene, while their progeny, mature circulating T cells, do. We clearly demonstrate the presence of the transgene in non-GFP-expressing as well as in GFP-expressing T cells using polymerase chain reaction (PCR). Based on these results, we suggest that the presence of a marker gene should be determined not only by the expression of the marker but also by analyzing the presence of the gene through DNA analysis.
In a recently held Keystone meeting, From Stem Cells to Therapy (Steamboat Springs, CO, 2003), several examples of the transgenic marking of stem cells to determine the presence of injected stem cells in a repaired organ that was damaged before injection were presented. It has been debated whether organ-specific stem cells can give rise to cells of other organs when injected in animals that have artificially damaged organs . The number of marked stem cells that can be traced in the repaired organ is rather low (in the worst case scenario, zero in thirteen million cells), raising doubt as to whether the transdifferentiation of stem cells is a likely event or only occurs in a very few instances . Cell fusion of muscle cells or hepatocytes with circulating stem cells could also explain the low number of marked cells . However, it remains to be explained how muscle cells that do not fuse with stem cells repair their damage.
Besides cell fusion, other mechanisms could explain the low number of marked cells in regenerated tissue. It has already been demonstrated that bone marrow stem cells do not replace damaged endothelial cells , indicating that more committed stem cells perform the initial repair. Likewise, when cardiotoxin is used to damage muscle cells in order to induce repair, only muscle cells are damaged and not the satellite cells . The latter cells are the immediate precursors of muscle cells. Both findings point to a mechanism in which damage is repaired by immediate precursor cells and not by the less committed stem cells, explaining the low number of marked cells in regenerated muscle. However, it remains to be demonstrated whether stem cells are able to replace satellite cells. Evidence is accumulating that serial long-term transplants do indeed give rise to bone marrow cell involvement in endothelial cell repair .
The other putative mechanism to explain the low number of marked cells in regenerated tissue is gene silencing. In transgenic experiments, stem cells are usually transfected with DNA that encodes a fluorescent protein, enabling easy detection of the fluorescent cells in tissue sections. Stem cells pass through a tremendous differentiation program to end up as cells of a specific organ. During this differentiation program, the chromatin also undergoes a tremendous epigenetic reprogramming to enable the transcription of genes needed at specific time points of differentiation. If the transgene used for marking ends up in a part of the chromatin that is packaged in the cell type studied, hardly any marked cells will be traced. An example of this was presented by A. M. Muller (University of Wurzburg) at the aforementioned meeting, in which neural stem cells could not give rise to hematopoietic progeny but did so after epigenetic reprogramming. Based on these considerations, marked cells should be identified by DNA technology as well, and not only by expression of the transgene. If the transgene is not present in the differentiated organ cell, then transdifferentiation is indeed a rare event.
To analyze the proposed silencing of transgenes, we isolated thymocytes from GFP transgenic mice to assess their GFP expression. As reported previously , GFP is expressed in eggs, demonstrating that the earliest cells express the transgene. As is evident from the flow cytometric profile in Figure 1, thymocytes of GFP transgenic mice hardly express GFP, while mature blood leukocytes do. Similar flow cytometric profiles were obtained for CD3+ thymocytes and CD3+ peripheral mononuclear cells, excluding the possibility that the difference in GFP expression was based on different cell types. We sorted the negative and positive thymocytes as indicated in Figure 1 and analyzed the presence of the transgene with PCR. The results are shown in Figure 2. It is clear that the transgene was present in non-GFP-expressing cells. Thymocytes are precursors of mature T cells and evidently have other parts of their genome silenced, compared with mature blood cells. An alternative explanation, that mRNA is less stable in thymocytes, can be ruled out because other GFP transgenic mice obtained from independent fertilizations and another integration site clearly showed green thymocytes in four of five cases .
Figure 1. Expression of GFP by leukocytes (A) and thymocytes (B) obtained from GFP transgenic mice (one example of six mice is depicted). The two markers (labeled N and M) indicate the cell populations that were sorted and used for subsequent DNA analyses.
Figure 2. DNA encoding GFP was found in the non-GFP-expressing as well as the GFP-expressing thymocytes. Lane 1: marker lane. Lanes 2 and 4: sorted non-GFP-expressing cells (two independent DNA isolations; gate N in Figure 1). Lanes 3 and 5: sorted GFP-expressing cells (two independent DNA isolations; gate M in Figure 1). Lane 6: control GFP plasmid. Lane 7: negative control.
In conclusion, we demonstrated that the tracking of differentiated marked stem cells should not only be based on the expression of the marker gene but also on the presence of the transgene as assessed by DNA methodology performed in parallel. Moreover, assessments of the involvement of stem cells in tissue repair should also be performed in studies in which the committed precursors are wiped out.
ACKNOWLEDGMENT
We thank A. Pennings, K. van Ginkel, and M. Looman for help with sorting and DNA analysis. We thank Dr. M. Okabe, University of Osaka, for providing us with the EGFP transgenic mice (http://133.1.15.131/TG/greenmouse.cfm). This study was supported by a grant from the Stichting Technische Wetenschappen, No. NGC 5611.
REFERENCES
Blau H, Brazelton T, Keshet G et al. Something in the eye of the beholder. Science 2002;298:361–363.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.
Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.
Hillebrands JL, Klatter FA, van Dijk WD et al. Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis. Nat Med 2002;8:194–195.
Couteaux R, Mira JC, d’Albis A. Regeneration of muscles after cardiotoxin injury. I. Cytological aspects. Biol Cell 1988;62:171–182.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Cogle CR, Wainman DA, Jorgensen ML et al. Adult human hematopoietic cells provide functional hemangioblast activity. Blood 2004;103:133–135.
Ikawa M, Yamada S, Nakanishi T et al. Green fluorescent protein (GFP) as a vital marker in mammals. Curr Top Dev Biol 1999;44:1–20.
Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.(Ruurd Torensma, Carl G. F)
Key Words. Stem cell differentiation ? Gene silencing ? Transgene expression ? EGFP marking ? Epigenetic reprogramming
Ruurd Torensma, Ph.D., Department of Tumorimmunology, UMC Nijmegen, Nijmegen Center for Molecular Life Sciences 187, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Telephone: 31-24-3610544; Fax: 31-24-3540339; e-mail: r.torensma@ncmls.kun.nl
Genetic marking of stem cells is a widely used method to trace transplanted cells in a nonmarked recipient. There is considerable debate as to whether organ-specific stem cells can give rise to cells of other organs (transdifferentiation). The number of marked hematopoietic stem cells that can be traced in a specific organ is rather low (less than one in one million cells), raising doubt as to whether the transdifferentiation of stem cells is a likely event or whether it only occurs in a very few instances. Cell fusion of damaged muscle cells with stem cells could also explain the low number of marked cells. Here, we demonstrate that expression of a marker gene, in this case, the gene encoding green fluorescent protein (GFP), is dependent on the differentiation status of the stem cell. T cells residing in the thymus of GFP transgenic mice do not express this transgene, while their progeny, mature circulating T cells, do. We clearly demonstrate the presence of the transgene in non-GFP-expressing as well as in GFP-expressing T cells using polymerase chain reaction (PCR). Based on these results, we suggest that the presence of a marker gene should be determined not only by the expression of the marker but also by analyzing the presence of the gene through DNA analysis.
In a recently held Keystone meeting, From Stem Cells to Therapy (Steamboat Springs, CO, 2003), several examples of the transgenic marking of stem cells to determine the presence of injected stem cells in a repaired organ that was damaged before injection were presented. It has been debated whether organ-specific stem cells can give rise to cells of other organs when injected in animals that have artificially damaged organs . The number of marked stem cells that can be traced in the repaired organ is rather low (in the worst case scenario, zero in thirteen million cells), raising doubt as to whether the transdifferentiation of stem cells is a likely event or only occurs in a very few instances . Cell fusion of muscle cells or hepatocytes with circulating stem cells could also explain the low number of marked cells . However, it remains to be explained how muscle cells that do not fuse with stem cells repair their damage.
Besides cell fusion, other mechanisms could explain the low number of marked cells in regenerated tissue. It has already been demonstrated that bone marrow stem cells do not replace damaged endothelial cells , indicating that more committed stem cells perform the initial repair. Likewise, when cardiotoxin is used to damage muscle cells in order to induce repair, only muscle cells are damaged and not the satellite cells . The latter cells are the immediate precursors of muscle cells. Both findings point to a mechanism in which damage is repaired by immediate precursor cells and not by the less committed stem cells, explaining the low number of marked cells in regenerated muscle. However, it remains to be demonstrated whether stem cells are able to replace satellite cells. Evidence is accumulating that serial long-term transplants do indeed give rise to bone marrow cell involvement in endothelial cell repair .
The other putative mechanism to explain the low number of marked cells in regenerated tissue is gene silencing. In transgenic experiments, stem cells are usually transfected with DNA that encodes a fluorescent protein, enabling easy detection of the fluorescent cells in tissue sections. Stem cells pass through a tremendous differentiation program to end up as cells of a specific organ. During this differentiation program, the chromatin also undergoes a tremendous epigenetic reprogramming to enable the transcription of genes needed at specific time points of differentiation. If the transgene used for marking ends up in a part of the chromatin that is packaged in the cell type studied, hardly any marked cells will be traced. An example of this was presented by A. M. Muller (University of Wurzburg) at the aforementioned meeting, in which neural stem cells could not give rise to hematopoietic progeny but did so after epigenetic reprogramming. Based on these considerations, marked cells should be identified by DNA technology as well, and not only by expression of the transgene. If the transgene is not present in the differentiated organ cell, then transdifferentiation is indeed a rare event.
To analyze the proposed silencing of transgenes, we isolated thymocytes from GFP transgenic mice to assess their GFP expression. As reported previously , GFP is expressed in eggs, demonstrating that the earliest cells express the transgene. As is evident from the flow cytometric profile in Figure 1, thymocytes of GFP transgenic mice hardly express GFP, while mature blood leukocytes do. Similar flow cytometric profiles were obtained for CD3+ thymocytes and CD3+ peripheral mononuclear cells, excluding the possibility that the difference in GFP expression was based on different cell types. We sorted the negative and positive thymocytes as indicated in Figure 1 and analyzed the presence of the transgene with PCR. The results are shown in Figure 2. It is clear that the transgene was present in non-GFP-expressing cells. Thymocytes are precursors of mature T cells and evidently have other parts of their genome silenced, compared with mature blood cells. An alternative explanation, that mRNA is less stable in thymocytes, can be ruled out because other GFP transgenic mice obtained from independent fertilizations and another integration site clearly showed green thymocytes in four of five cases .
Figure 1. Expression of GFP by leukocytes (A) and thymocytes (B) obtained from GFP transgenic mice (one example of six mice is depicted). The two markers (labeled N and M) indicate the cell populations that were sorted and used for subsequent DNA analyses.
Figure 2. DNA encoding GFP was found in the non-GFP-expressing as well as the GFP-expressing thymocytes. Lane 1: marker lane. Lanes 2 and 4: sorted non-GFP-expressing cells (two independent DNA isolations; gate N in Figure 1). Lanes 3 and 5: sorted GFP-expressing cells (two independent DNA isolations; gate M in Figure 1). Lane 6: control GFP plasmid. Lane 7: negative control.
In conclusion, we demonstrated that the tracking of differentiated marked stem cells should not only be based on the expression of the marker gene but also on the presence of the transgene as assessed by DNA methodology performed in parallel. Moreover, assessments of the involvement of stem cells in tissue repair should also be performed in studies in which the committed precursors are wiped out.
ACKNOWLEDGMENT
We thank A. Pennings, K. van Ginkel, and M. Looman for help with sorting and DNA analysis. We thank Dr. M. Okabe, University of Osaka, for providing us with the EGFP transgenic mice (http://133.1.15.131/TG/greenmouse.cfm). This study was supported by a grant from the Stichting Technische Wetenschappen, No. NGC 5611.
REFERENCES
Blau H, Brazelton T, Keshet G et al. Something in the eye of the beholder. Science 2002;298:361–363.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.
Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.
Hillebrands JL, Klatter FA, van Dijk WD et al. Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis. Nat Med 2002;8:194–195.
Couteaux R, Mira JC, d’Albis A. Regeneration of muscles after cardiotoxin injury. I. Cytological aspects. Biol Cell 1988;62:171–182.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Cogle CR, Wainman DA, Jorgensen ML et al. Adult human hematopoietic cells provide functional hemangioblast activity. Blood 2004;103:133–135.
Ikawa M, Yamada S, Nakanishi T et al. Green fluorescent protein (GFP) as a vital marker in mammals. Curr Top Dev Biol 1999;44:1–20.
Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.(Ruurd Torensma, Carl G. F)