scholarly journals Increased Longevity of Continuous Bone Marrow Cultures and Radioresistance of Bone Marrow Stromal Cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5679-5679
Author(s):  
Andrew Henderson ◽  
Renee Fisher ◽  
Michael Epperly ◽  
Donna Shields ◽  
Lora Rigatti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Marrow Transplant ◽  
Marrow Stromal Cell ◽  
Free Interval ◽  
Bone Marrow Cultures ◽  
Total Body ◽  
Marrow Cultures

Abstract Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss. Materials & Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis. Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466). Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748). Conclusions: The results showing increased longevity of hematopoiesis in LTBMCs of marrow from mice displaying an absence of SOD1 and the radioresistance of derived bone marrow stromal cell lines represent two unexpected pleiotrophic effects of the SOD1 G93A genotype. Further studies will be required to determine how marrow transplant after TBI prolonged the paralysis free interval in these ALS mice. Disclosures No relevant conflicts of interest to declare.

Biology of Marrow Stromal Cell Lines Derived from Long-Term Bone Marrow Cultures of Trp53-Deficient Mice

1999 ◽  
Vol 152 (1) ◽  
pp. 29 ◽  
Author(s):  
Michael W. Epperly ◽  
Jenifer A. Bray ◽  
Timothy M. Carlos ◽  
Edward Prochownik ◽  
Joel S. Greenberger
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Marrow Stromal Cell ◽  
Bone Marrow Cultures ◽  
Deficient Mice ◽  
Marrow Cultures

scholarly journals Transformed Phenotype of Bone Marrow Stromal Cell Lines Derived from K14E7 Fancd2-/- mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4795-4795
Author(s):  
Aranee Sivanathan ◽  
Xichen Zhang ◽  
Darcy Franicola ◽  
Shaonan Cao ◽  
Donna Shields ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Wild Type ◽  
Marrow Stromal Cell ◽  
Cytokeratin 14 ◽  
E7 Oncogene ◽  
Marrow Cultures

Abstract To determine whether cytokeratin 14 promoter linked expression of the Human Papilloma Virus (HPV) oncogene detectably influenced biologic parameters of cell phenotypes other than squamous epithelium, continuous bone marrow cultures were derived from K14E7 Fancd2-/- mice (Park, et al., Cancer Research, 70(23): 9959-9968, 2010). Long-term bone marrow cultures derived from K14E7 Fancd2-/-, control Fancd2-/- (129/Sv), K14E7 (FVB/N), and wild type 129/Sv X FVB/N F1 mice were evaluated for longevity of hematopoiesis in long-term cultures and stromal cell lines were derived from each. Similar to Fancd2-/- mouse, long-term marrow cultures, K14E7 Fancd2-/- marrow cultures demonstrated decreased longevity of hematopoiesis with cessation of production of multi-lineage colony forming progenitor cells after 14 weeks. In contrast, wild type F1 and K14E7 long-term marrow cultures continued to produce hematopoietic cells for a significantly longer duration 25 weeks (p=0.0257). Bone marrow stromal and IL-3 dependent hematopoietic cell lines were derived from each genotype marrow culture. K14E7 Fancd2-/- hematopoietic cells showed reversal of the radiation resistance of Fancd2-/- IL-3 dependent cell lines (D0 of 1.34 ± 0.197Gy, ñ 4.0 ± 0.9 compared to D02.213 ± 0.124 Gy (p = 0.0284), ñ 3.3 ± 0.8. Thus, one phenotypic difference associated with K14E7 oncogene expression was reversal of radioresistance of Fancd2-/- hematopoietic cells. In contrast, bone marrow stromal cell lines from K14E7 Fancd2-/- remained radiosensitive similar to those from Fancd2-/- mice. K14E7 and wild type F1 marrow stromal cell lines showed intermediate radioresistance (p= 0.1759). To determine whether the E6/E7 oncogene had a biological effect in tissues other than squamous epithelium, tissue analysis for cytokeratins 13, 14, 6, and 10 was carried out. Cytokeratin 14 was detected only in squamous cells of the esophagus and oral cavity, not in bone marrow. E6/E7 oncogene was detected only in squamous cell lines expressing cytokeratin 14. However, bone marrow stromal cell lines from K14E7 Fancd2-/- marrow cultures demonstrated a unique phenomenon of cellular density, piling up and formation of tumors in vitro. Each of 22 single cell derived clonal sub-lines of K14E7 Fancd2-/- stromal cell lines demonstrated the same transformed phenotype. These data provide support for indirect effects of the E7 oncogene linked to the K14 promoter in Fancd2-/- hematopoietic and mesenchymal stem cell tissues. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

scholarly journals Radiation Resistance of Double Knockout (DKO) Smad3-/- Fancd2-/- (129/Sv) Mouse Bone Marrow Stromal Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3901-3901
Author(s):  
Kevin Keppel ◽  
Michael W. Epperly ◽  
Donna Shields ◽  
Wen Hou ◽  
Darcy Franicola ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Mouse Strains ◽  
Double Knockout ◽  
Marrow Stromal Cell ◽  
Marrow Cultures

Abstract Introduction: Bone marrow progenitor cells from Fanconi Anemia (FA) patients have a hyperactive TGF-β signaling pathway which may explain hematopoietic stem cell depletion leading to bone marrow failure (Zhang, et al, Cell Stem Cell, 18:668-681, 2016). To determine whether blockade of TGF-B signaling by knockout of Smad3 normalized hematopoiesis in Fancd2-/- mice, we bred two double knockout mouse strains. The first (DKO#1) was derived by crossing C57BL/6 Fancd2 +/- mice with 129/Sv Smad3 +/- mice. The second (DKO#2) was derived by mating 129/Sv Fancd2 +/- mice with the same 129/Sv Smad3 +/- mice. Materials and Methods: Long term bone marrow cultures (LTBMCs) were established from DKO#1, DKO#2, parental mouse strains and F1 (129/Sv X C57Bl/6) control mice for DKO#1, 129/Sv Smad3 -/-, and C57BL/6 Smad3 -/- mice . The cultures were scored for weekly numbers of cobblestone islands as an indicator of stem cells in the adherent layer, weekly production of nonadherent cells, and cells producing Day 7 and Day 14 CFU-GEMM .Bone marrow stromal cell lines were derived from the adherent layer of LTBMCs and radiosensitivity measured in clonogenic radiation survival curves. Cells were irradiated to doses of 0 to 8 Gy, plated in 4 well linbro plates, incubated for 7 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted. Western analysis of the cell lines quantitated expression levels of proteins involved in the TGF-β signaling pathway, and DNA double strand break repair by homologous recombination and nonhomologous end-joining. Results: Both DKO1 and DKO2 LTBMCs showed decreased duration and magnitude of hematopoiesis, thus being similar to that observed with both C57BL/6 Fancd2-/- and 129/Sv Fancd2-/- mouse marrow cultures. Both were significantly lower than that for control mouse or Smad3-/- (129/Sv) marrow cultures. As expected, radiation survival curves showed that both C57Bl/6 Fancd2-/- and 129/Sv Fancd2-/- marrow stromal cell lines were radiosensitive compared to control cell lines including: F1 control, C57BL/6 control, and 129/Sv control (Berhane, et al, Rad Res. 181:76-89, 2014, Berhane et.al, Rad Res 182:35-49, 2014). In contrast marrow stromal cell lines from Smad3-/- (129/Sv) marrow cultures were radioresistant (Epperly, et al, Rad Res 165:671-677, 2006). Fresh marrow CFU-GEMM from both DKO#1 and DKO#2 mice showed resistance to abrogation of colony formation by increasing concentrations of TGF-B, (similar to the 129/Sv Smad3-/- cell line). In contrast, cell lines from all controls and both Fancd2-/- mice showed clear TGF-B mediated inhibition of hemopoietic colony formation. In striking contrast to the above similarities between DKO#1 and DKO#2 mice, marrow stromal cell lines from DKO#1 were radiosensitive (like theirFancd2-/- parent) while those from DKO#2 were radioresistant (like their Smad3-/- parent). Thus, DKO#1 retained the C57Bl/6 Fancd2-/- genotype cell line radiosensitivity: Do of 1.41 ± 0.03 Gy and 1.45 ± 0.05 Gy respectively, and were more radiosensitive than the control F1 bone marrow stromal cell line (p = 0.0230 and 0.0418, respectively) and DKO#2 retained the 129/Sv Smad3-/- cell line genotype radioresistance (Do = 2.15 ± 0.13 Gy) compared to control 129/Sv stromal cells (Do = 1.86 ± 0.04, p = 0.0054). Western analysis revealed that p21 was elevated in DKO#2 but not DKO#1 marrow stromal cell lines. Conclusions: While marrow from both DKO#1 and DKO#2 mice showed resistance to TGF-B signaling consistent with their smad3-/- genotype, only DKO#1 stromal cells retained the radiosensitivity of their Fancd2-/- genotype. Reduced p21 in irradiated DKO#1 marrow stromal cells may have allowed procession through G to S phase causing reduced time for DNA repair and radiosensitivity. The irradiated DKO#2 cells may have been blocked by p21 from passing through the G1 checkpoint and may have allowed DNA strand break repair and radioresistance. These DKO mice and derived cell lines should be valuable for analysis of the interaction of TGF-B signaling and FA pathways. Supported by NIAID/NIH U19-AI068021 Disclosures No relevant conflicts of interest to declare.

Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule

Cell ◽  
1987 ◽  
Vol 48 (6) ◽  
pp. 1009-1021 ◽  
Author(s):  
Cheryl A. Whitlock ◽  
George F. Tidmarsh ◽  
Christa Muller-Sieburg ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Marrow Stromal Cell

Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors

1993 ◽  
Vol 54 (6) ◽  
pp. 1017-1021 ◽  
Author(s):  
Sigrun Gabius ◽  
Ralf Wawotzny ◽  
Sabine Wilholm ◽  
Ulrikc Martin ◽  
Bernhard Wörmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Marrow Stromal Cell ◽  
Human Lymphoid Cell ◽  
Cell Layers ◽  
Sugar Receptors ◽  
Lymphoid Cell Lines

scholarly journals Expression of the c-kitligand and interleukin 6 genes in mouse bone marrow stromal cell lines

Stem Cells ◽  
1994 ◽  
Vol 12 (4) ◽  
pp. 409-415 ◽  
Author(s):  
Takeshi Otsuka ◽  
Tomonori Ogo ◽  
Teruaki Nakano ◽  
Hiroaki Niiro ◽  
Seiji Kuga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Mouse Bone Marrow ◽  
Marrow Stromal Cell

Expression of the Smad3 Transgene Restores Radiosensitivity and Migratory Capacity to a Smad3−/− Clonal Bone Marrow Stromal Cell Line.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4307-4307
Author(s):  
Michael W. Epperly ◽  
Shaonan Cao ◽  
Xichen Zhang ◽  
Emily E. Greenberger ◽  
Julie Goff ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Gene Product ◽  
Cell Lines ◽  
Stromal Cell ◽  
Cell Tracking ◽  
Bone Marrow Stromal Cell ◽  
Saturation Density ◽  
Marrow Stromal Cell ◽  
Migratory Capacity

Abstract An intact Smad3 gene product is critical for a functioning signal transduction pathway following TGF binding to the TGF-β receptor. We have previously established Smad3−/− and Smad3+/+ long term bone marrow cultures (LTBMCs) and isolated clonal bone marrow stromal cell lines from each. The Smad3−/− cells were smaller in size but had a faster cell doubling time (24 hours compared to 48 hours) and increased saturation density compared to +/+ cells (15.3 ± 1.0 x 105 cells/25 mm2 flask compared to 3.8 ± 0.1 x 105, p = 0.003). The plating efficiency of the lines was similar (18.3 ± 2.7 compared to 15.5 ± 1.7, p = 0.417). We transfected the Smad3−/− cell line with a retrovirus containing the Smad3 transgene, and selected a subclone expressing the transgene mRNA, designated Smad3−/−(3). Smad3−/−(3) cells were increased in size to that of Smad3+/+ cells, and showed decreased cell saturation density. Using the Cytoworks computer controlled cell tracking Bioreactor, we measured the migration of each clonal line. Tissue culture wells of 100 cells per well were followed for 5 days tracking each cell in quadruplicate wells per cell line. Smad3+/+ cells migrated significantly faster over 5 days in culture compared to Smad3−/− cells. (The average velocities were 0.62 μm/min for Smad3+/+ and 0.36 μm/min for Smad3−/−, p&lt;0.0001). Over 5 days, the average velocities for Smad3+/+ cells were 0.51, 0.51, 0.52, 0.72, 0.91 μm/min, and for Smad3−/− cells were 0.28, 0.38, 0.41, 0.37, 0.35 μm/min. The 5 p-values comparing these cell lines were all &lt;0.0001. The 7 day clonagenic irradiation survival curve showed that Smad3+/+ and Smad3−/−(3) cells were significantly more sensitive (D0 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− cell line (D0 = 2.43 ± 0.06 Gy, p=0.0016 and 0.0103). These results demonstrate a concordance of radioresistance and decreased migratory capacity in bone marrow stromal cells devoid of a functioning Smad3 gene product, and restoration of both properties following overexpression of the transgene product. These data may help explain the decreased radiation fibrosis observed in Smad3−/− mice.

Sign in / Sign up

Export Citation Format

Share Document