title: Novel daidzein analogs enhance osteogenic activity of bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells through estrogen receptor dependent and independent mechanisms.
abstract: BACKGROUND
  Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein and daidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs). Herein, the effects of daidzein analogs on the osteogenic differentiation efficiency of human BMSC and adipose-derived stromal/stem cells (ASC) were explored.
  
  
  METHODS
  BMSCs and ASCs underwent osteogenic differentiation in the presence of vehicle, 17β-estradiol (E2), phytoestrogens, or daidzein analogs. Cells were stained for alkaline phosphatase (ALP) enzymatic activity, calcium deposition by alizarin red s, and phosphate mineralization by silver nitrate. Gene expression analysis was conducted on cells treated with daidzein analogs.
  
  
  RESULTS
  Cells treated with E2, daidzein, or genistein increased calcium deposition by 1.6-, 1.5-, and 1.4-fold, respectively, relative to vehicle-treated BMSCs and 1.6-, 1.7-, and 1.4-fold relative to vehicle-treated ASCs, respectively. BMSCs treated with daidzein analog 2c, 2g, and 2l demonstrated a 1.6-, 1.6-, and 1.9-fold increase in calcium deposition relative to vehicle-treated BMSCs, respectively, while ASCs treated with daidzein analog 2c, 2g, or 2l demonstrated a 1.7-, 2.0-, and 2.2-fold increase in calcium deposition relative to vehicle-treated ASCs, respectively. Additional analysis with BMSCs and ASCs was conducted in the more efficient compounds: 2g and 2l. ALP activity and phosphate mineralization was increased in 2g- and 2l-treated cells. The analysis of lineage specific gene expression demonstrated increased expression of key osteogenic genes (RUNX2, c-FOS, SPARC, DLX5, SPP1, COL1A1, IGF1, SOST, and DMP1) and earlier induction of these lineage specific genes, following treatment with 2g or 2l, relative to vehicle-treated cells. Estrogen receptor (ER) inhibitor studies demonstrated that ER antagonist fulvestrant inhibited the osteogenic differentiation of 2g in BMSCs and ASCs, while fulvestrant only attenuated the effects of 2l, suggesting that 2l acts by both ER dependent and independent pathways.
  
  
  CONCLUSIONS
  These studies provide support for exploring the therapeutic efficacy of daidzein derivatives for the treatment of osteoporosis. Furthermore, the patterns of gene induction differed following treatment with each daidzein analog, suggesting that these daidzein analogs activate distinct ER and non-ER pathways to induce differentiation in BMSCs and ASCs.
authors.affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. alin1@tulane.edu.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Strong
authors.affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. johlstei@tulane.edu.
authors.forename: Jason F
authors.initials: JF
authors.lastname: Ohlstein
authors.affiliation: Department of Chemistry and RCMI Cancer Research Program, Xavier University of Louisiana, New Orleans, LA, 70125, USA. qjiang1@my.uno.edu.
authors.forename: Quan
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: Department of Chemistry and RCMI Cancer Research Program, Xavier University of Louisiana, New Orleans, LA, 70125, USA. qzhang1@xula.edu.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and RCMI Cancer Research Program, Xavier University of Louisiana, New Orleans, LA, 70125, USA. szheng@xula.edu.
authors.forename: Shilong
authors.initials: S
authors.lastname: Zheng
authors.affiliation: US Department of Agriculture, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, LA, 70124, USA. steve.boue@ars.usda.gov.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Boue
authors.affiliation: Department of Medicine, Tulane University School of Medicine, New Orleans, LA, 70112, USA. selliot@tulane.edu.
authors.forename: Steven
authors.initials: S
authors.lastname: Elliott
authors.affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. jgimble@tulane.edu.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Gimble
authors.affiliation: Department of Medicine, Tulane University School of Medicine, New Orleans, LA, 70112, USA. mburow@tulane.edu.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Burow
authors.affiliation: Department of Chemistry and RCMI Cancer Research Program, Xavier University of Louisiana, New Orleans, LA, 70125, USA. gwang@xula.edu.
authors.forename: Guangdi
authors.initials: G
authors.lastname: Wang
authors.affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. bbunell@tulane.edu.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Bunnell
mesh_terms: D053687:Adult Stem Cells / Q000187:drug effects / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D050071:Bone Density Conservation Agents / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D000077267:Fulvestrant; D006801:Humans; D020128:Inhibitory Concentration 50; D007529:Isoflavones / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010012:Osteogenesis; D010024:Osteoporosis / Q000188:drug therapy; D048789:Phytoestrogens / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000502:physiology*
chemical_list: D050071:Bone Density Conservation Agents; D007529:Isoflavones; D048789:Phytoestrogens; D011960:Receptors, Estrogen; D000077267:Fulvestrant; C013908:daidzin; D004958:Estradiol; D000469:Alkaline Phosphatase