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Precancerous stem cells have the potential for both benign and malignant differentiation.

Chen L, Shen R, Ye Y, Pu XA, Liu X, Duan W, Wen J, Zimmerer J, Wang Y, Liu Y, Lasky LC, Heerema NA, Perrotti D, Ozato K, Kuramochi-Miyagawa S, Nakano T, Yates AJ, Carson WE, Lin H, Barsky SH, Gao JX - PLoS ONE (2007)

Bottom Line: However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized.Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2.Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.

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Related in: MedlinePlus

pCSCs can differentiate into various type of tissue cells.A, Differentiation of pCSCs into hematopoietic and non-hematopoietic cells: The lethally irradiated CD45.1 congenic B6 mice were injected i.v. with 1×106 2C4 (n = 5) or eGFP+ 2C4G2 cells (n = 10), along with 5×105 recipient-type BM cells, as described above. The mice were sacrificed 5 months post transfer. Various organs, including the liver, kidney, spleen and adipose tissues were harvested, fixed in 10% formaldehyde of PBS, prepared for H & E. staining, and examined under fluorescent microscope. At least three discontinuous sections (100 µm/step) were examined for each organ to ensure that eGFP+ cells were identified under the fluorescent microscope. The morphology of eGFP+ cells was determined under the bright field of the fluorescent microscope (original magnification ×1000). B–E, Development of pCSCs in blastocyst chimeric mice: E3.5 dpc of FVB mice were injected with 2C4G2 (8∼10 cells per blastocyst) and transferred to pseudopregnant surrogate mothers. The progeny were delivered and grew to adult without any complication. The data shown are from one of two experiments in which 8 progeny (male: n = 6; female: n = 2) were obtained. One male mouse died of fighting at 3 months of age. B, eGFP+ RBCs in 7/8 of the chimeric mice: The data represent the air-dried blood smears from two mice, at the age of 2 months, examined under, respectively, the bright and fluorescent fields of a fluorescent microscope (Nike, E400, Japan). C, pCSC-derived eGFP+CD45+ cells: peripheral blood was harvested from the chimeric mice at age 2 months (n = 6; other two pregnant mice were not examined) or control FVB mice (n = 10), stained with PE-conjugated mAb to CD45, and analyzed by flow cytometry. D & E, Living image of the chimeric mice: A representative living image of the chimeric mice at 4 months of age is shown in D, demonstrated by IVIS imaging systems incorporated with Living Imaging® software (Xenogen Inc.); and the eGFP-derived photon counts in the region of interest (ROI) of 7 mice are shown in E. Normal FVB mice were used as control for living imaging.
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pone-0000293-g003: pCSCs can differentiate into various type of tissue cells.A, Differentiation of pCSCs into hematopoietic and non-hematopoietic cells: The lethally irradiated CD45.1 congenic B6 mice were injected i.v. with 1×106 2C4 (n = 5) or eGFP+ 2C4G2 cells (n = 10), along with 5×105 recipient-type BM cells, as described above. The mice were sacrificed 5 months post transfer. Various organs, including the liver, kidney, spleen and adipose tissues were harvested, fixed in 10% formaldehyde of PBS, prepared for H & E. staining, and examined under fluorescent microscope. At least three discontinuous sections (100 µm/step) were examined for each organ to ensure that eGFP+ cells were identified under the fluorescent microscope. The morphology of eGFP+ cells was determined under the bright field of the fluorescent microscope (original magnification ×1000). B–E, Development of pCSCs in blastocyst chimeric mice: E3.5 dpc of FVB mice were injected with 2C4G2 (8∼10 cells per blastocyst) and transferred to pseudopregnant surrogate mothers. The progeny were delivered and grew to adult without any complication. The data shown are from one of two experiments in which 8 progeny (male: n = 6; female: n = 2) were obtained. One male mouse died of fighting at 3 months of age. B, eGFP+ RBCs in 7/8 of the chimeric mice: The data represent the air-dried blood smears from two mice, at the age of 2 months, examined under, respectively, the bright and fluorescent fields of a fluorescent microscope (Nike, E400, Japan). C, pCSC-derived eGFP+CD45+ cells: peripheral blood was harvested from the chimeric mice at age 2 months (n = 6; other two pregnant mice were not examined) or control FVB mice (n = 10), stained with PE-conjugated mAb to CD45, and analyzed by flow cytometry. D & E, Living image of the chimeric mice: A representative living image of the chimeric mice at 4 months of age is shown in D, demonstrated by IVIS imaging systems incorporated with Living Imaging® software (Xenogen Inc.); and the eGFP-derived photon counts in the region of interest (ROI) of 7 mice are shown in E. Normal FVB mice were used as control for living imaging.

Mentions: To test these two possibilities, we stably transduced 2C4 cells with lentiviruses, which carried an enhanced green fluorescent protein (eGFP) gene, to track the fate of pCSCs in recipients. We obtained several clones, such as clone 2C4G2 (Fig. S4) that expressed a high level of eGFP. The 2C4G2 and parent 2C4 cells were intravenously transplanted, respectively, into lethally irradiated, BM-reconstituted CD45.1 congenic mice, which should provide stem cells with a vigorously regenerative environmental cue. The donor-derived eGFP+ cells, albeit lower in frequency, were readily detected in various organs, such as the spleen, liver, kidney, small intestine, or adipose tissues, of all the mice that had received 2C4G2, but not 2C4 cells, for 5 months (Fig. 3a and data not shown). Some eGFP+ cells exhibited the morphology of tissue origin, including endothelial cells, tubular epithelial cells, Kupffer's cells, histiocytes, macrophages/monocytes, and hepatoid cells (Fig. 3a). In the liver, eGFP+ Kupffer's cells and hepatoid cells were usually found in the regenerative areas (Fig. 3a). Interestingly, none of the observed eGFP+ cells exhibited significant dysplastic changes (Fig. 3a). The results suggest that while the pCSCs were generally unable to completely differentiate into mature hematopoietic cells in vitro (Fig. S1 & S2); some of them may differentiate into tissue-specific cells in a regenerative environment. Interestingly, none of the recipients developed tumors under the conditions.


Precancerous stem cells have the potential for both benign and malignant differentiation.

Chen L, Shen R, Ye Y, Pu XA, Liu X, Duan W, Wen J, Zimmerer J, Wang Y, Liu Y, Lasky LC, Heerema NA, Perrotti D, Ozato K, Kuramochi-Miyagawa S, Nakano T, Yates AJ, Carson WE, Lin H, Barsky SH, Gao JX - PLoS ONE (2007)

pCSCs can differentiate into various type of tissue cells.A, Differentiation of pCSCs into hematopoietic and non-hematopoietic cells: The lethally irradiated CD45.1 congenic B6 mice were injected i.v. with 1×106 2C4 (n = 5) or eGFP+ 2C4G2 cells (n = 10), along with 5×105 recipient-type BM cells, as described above. The mice were sacrificed 5 months post transfer. Various organs, including the liver, kidney, spleen and adipose tissues were harvested, fixed in 10% formaldehyde of PBS, prepared for H & E. staining, and examined under fluorescent microscope. At least three discontinuous sections (100 µm/step) were examined for each organ to ensure that eGFP+ cells were identified under the fluorescent microscope. The morphology of eGFP+ cells was determined under the bright field of the fluorescent microscope (original magnification ×1000). B–E, Development of pCSCs in blastocyst chimeric mice: E3.5 dpc of FVB mice were injected with 2C4G2 (8∼10 cells per blastocyst) and transferred to pseudopregnant surrogate mothers. The progeny were delivered and grew to adult without any complication. The data shown are from one of two experiments in which 8 progeny (male: n = 6; female: n = 2) were obtained. One male mouse died of fighting at 3 months of age. B, eGFP+ RBCs in 7/8 of the chimeric mice: The data represent the air-dried blood smears from two mice, at the age of 2 months, examined under, respectively, the bright and fluorescent fields of a fluorescent microscope (Nike, E400, Japan). C, pCSC-derived eGFP+CD45+ cells: peripheral blood was harvested from the chimeric mice at age 2 months (n = 6; other two pregnant mice were not examined) or control FVB mice (n = 10), stained with PE-conjugated mAb to CD45, and analyzed by flow cytometry. D & E, Living image of the chimeric mice: A representative living image of the chimeric mice at 4 months of age is shown in D, demonstrated by IVIS imaging systems incorporated with Living Imaging® software (Xenogen Inc.); and the eGFP-derived photon counts in the region of interest (ROI) of 7 mice are shown in E. Normal FVB mice were used as control for living imaging.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808425&req=5

pone-0000293-g003: pCSCs can differentiate into various type of tissue cells.A, Differentiation of pCSCs into hematopoietic and non-hematopoietic cells: The lethally irradiated CD45.1 congenic B6 mice were injected i.v. with 1×106 2C4 (n = 5) or eGFP+ 2C4G2 cells (n = 10), along with 5×105 recipient-type BM cells, as described above. The mice were sacrificed 5 months post transfer. Various organs, including the liver, kidney, spleen and adipose tissues were harvested, fixed in 10% formaldehyde of PBS, prepared for H & E. staining, and examined under fluorescent microscope. At least three discontinuous sections (100 µm/step) were examined for each organ to ensure that eGFP+ cells were identified under the fluorescent microscope. The morphology of eGFP+ cells was determined under the bright field of the fluorescent microscope (original magnification ×1000). B–E, Development of pCSCs in blastocyst chimeric mice: E3.5 dpc of FVB mice were injected with 2C4G2 (8∼10 cells per blastocyst) and transferred to pseudopregnant surrogate mothers. The progeny were delivered and grew to adult without any complication. The data shown are from one of two experiments in which 8 progeny (male: n = 6; female: n = 2) were obtained. One male mouse died of fighting at 3 months of age. B, eGFP+ RBCs in 7/8 of the chimeric mice: The data represent the air-dried blood smears from two mice, at the age of 2 months, examined under, respectively, the bright and fluorescent fields of a fluorescent microscope (Nike, E400, Japan). C, pCSC-derived eGFP+CD45+ cells: peripheral blood was harvested from the chimeric mice at age 2 months (n = 6; other two pregnant mice were not examined) or control FVB mice (n = 10), stained with PE-conjugated mAb to CD45, and analyzed by flow cytometry. D & E, Living image of the chimeric mice: A representative living image of the chimeric mice at 4 months of age is shown in D, demonstrated by IVIS imaging systems incorporated with Living Imaging® software (Xenogen Inc.); and the eGFP-derived photon counts in the region of interest (ROI) of 7 mice are shown in E. Normal FVB mice were used as control for living imaging.
Mentions: To test these two possibilities, we stably transduced 2C4 cells with lentiviruses, which carried an enhanced green fluorescent protein (eGFP) gene, to track the fate of pCSCs in recipients. We obtained several clones, such as clone 2C4G2 (Fig. S4) that expressed a high level of eGFP. The 2C4G2 and parent 2C4 cells were intravenously transplanted, respectively, into lethally irradiated, BM-reconstituted CD45.1 congenic mice, which should provide stem cells with a vigorously regenerative environmental cue. The donor-derived eGFP+ cells, albeit lower in frequency, were readily detected in various organs, such as the spleen, liver, kidney, small intestine, or adipose tissues, of all the mice that had received 2C4G2, but not 2C4 cells, for 5 months (Fig. 3a and data not shown). Some eGFP+ cells exhibited the morphology of tissue origin, including endothelial cells, tubular epithelial cells, Kupffer's cells, histiocytes, macrophages/monocytes, and hepatoid cells (Fig. 3a). In the liver, eGFP+ Kupffer's cells and hepatoid cells were usually found in the regenerative areas (Fig. 3a). Interestingly, none of the observed eGFP+ cells exhibited significant dysplastic changes (Fig. 3a). The results suggest that while the pCSCs were generally unable to completely differentiate into mature hematopoietic cells in vitro (Fig. S1 & S2); some of them may differentiate into tissue-specific cells in a regenerative environment. Interestingly, none of the recipients developed tumors under the conditions.

Bottom Line: However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized.Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2.Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors-namely, precancerous stem cells (pCSCs) -have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.

Show MeSH
Related in: MedlinePlus