Endogenous Erythroid and Megakaryocytic Colony Formation in Serum-Free, Cytokine-Free Collagen Gels

Background/Aims: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. Methods: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. Results: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. Conclusion: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

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The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture

AJP Cell Physiology ◽

10.1152/ajpcell.00168.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. C1396-C1407 ◽

Cited By ~ 95

Author(s):

K. M. Howson ◽

A. C. Aplin ◽

M. Gelati ◽

G. Alessandri ◽

E. A. Parati ◽

...

Keyword(s):

Smooth Muscle ◽

Progenitor Cells ◽

Rat Aorta ◽

Mesenchymal Cells ◽

Serum Free Medium ◽

Collagen Gels ◽

Free Medium ◽

Culture Dish ◽

Serum Free ◽

Cell Markers

Pericytes play an important role in modulating angiogenesis, but the origin of these cells is poorly understood. To evaluate whether the mature vessel wall contains pericyte progenitor cells, nonendothelial mesenchymal cells isolated from the rat aorta were cultured in a serum-free medium optimized for stem cells. This method led to the isolation of anchorage-independent cells that proliferated slowly in suspension, forming spheroidal colonies. This process required basic fibroblast growth factor (bFGF) in the culture medium, because bFGF withdrawal caused the cells to attach to the culture dish and irreversibly lose their capacity to grow in suspension. Immunocytochemistry and RT-PCR analysis revealed the expression of the precursor cell markers CD34 and Tie-2 and the absence of endothelial cell markers (CD31 and endothelial nitric oxide synthase, eNOS) and smooth muscle cell markers (α-smooth muscle actin, α-SMA). In addition, spheroid-forming cells were positive for NG2, nestin, PDGF receptor (PDGFR)-α, and PDGFR-β. Upon exposure to serum, these cells lost CD34 expression, acquired α-SMA, and attached to the culture dish. Returning these cells to serum-free medium failed to restore their original spheroid phenotype, suggesting terminal differentiation. When embedded in collagen gels, spheroid-forming cells rapidly migrated in response to PDGF-BB and became dendritic. Spheroid-forming cells cocultured in collagen with angiogenic outgrowths of rat aorta or isolated endothelial cells transformed into pericytes. These results demonstrate that the rat aorta contains primitive mesenchymal cells capable of pericyte differentiation. These immature cells may represent an important source of pericytes during angiogenesis in physiological and pathological processes. They may also provide a convenient supply of mural cells for vascular bioengineering applications.

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Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture

Blood ◽

10.1182/blood.v79.11.2855.2855 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2855-2860 ◽

Cited By ~ 105

Author(s):

K Tsuji ◽

SD Lyman ◽

T Sudo ◽

SC Clark ◽

M Ogawa

Keyword(s):

Colony Formation ◽

Serum Free ◽

Steel Factor ◽

Gm Csf ◽

Synergistic Interactions ◽

Two Factors ◽

Factor Combination ◽

Hematopoietic Factors ◽

Interleukin 11 ◽

Stimulating Factor

Abstract Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin- 6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL- 11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two- factor combination in serum-free culture was SF plus IL-3. In serum- free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.

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Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture

Blood ◽

10.1182/blood.v79.11.2855.bloodjournal79112855 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2855-2860 ◽

Cited By ~ 1

Author(s):

K Tsuji ◽

SD Lyman ◽

T Sudo ◽

SC Clark ◽

M Ogawa

Keyword(s):

Colony Formation ◽

Serum Free ◽

Steel Factor ◽

Gm Csf ◽

Synergistic Interactions ◽

Two Factors ◽

Factor Combination ◽

Hematopoietic Factors ◽

Interleukin 11 ◽

Stimulating Factor

Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin- 6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL- 11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two- factor combination in serum-free culture was SF plus IL-3. In serum- free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.

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