Differentiated adipose-derived stem cell cocultures for bone regeneration in polymer scaffolds in vivo.

Critical-sized bone defects can lead to significant morbidity, and interventions are limited by the availability and donor-site morbidity of bone grafts. Polymer scaffolds seeded with cells have been explored to replace bone grafts. Adipose-derived stem cells have shown great promise for vascularization and osteogenesis of these constructs, and cocultures of differentiated stem cells are being explored to augment vessel and bone formation. Adipose-derived stem cells were differentiated into endothelial cells and osteoblasts, and in vitro studies showed increased proliferation of cocultured cells compared with undifferentiated adipose-derived stem cells and monocultures of endothelial cells and osteoblasts. The cells were seeded into polylactic acid gas-plasma-treated scaffolds as cocultures and monocultures and then implanted into critical-sized rat calvarial defects. The cocultures were in a 1:1 osteoblast to endothelial cell ratio. The increase in proliferation seen by the cocultured cells in vitro did not translate to increased vascularization and osteogenesis in vivo. In vivo, there were trends of increased vascularization in the endothelial cell group and increased osteogenesis in the osteoblast and endothelial monoculture groups, but no increase was seen in the coculture group compared with the undifferentiated adipose-derived stem cells. Endothelial cells enhance vascularization and osteoblast and endothelial cell monocultures enhance bone formation in the polymer scaffold. Predifferentiation of adipose-derived stem cells is promising for improving vascularization and osteogenesis in polymer scaffolds but requires future evaluation of coculture ratios to fully characterize this response

Infusion of Some but Not All Types of Human Perinatal Stromal Cells Prevent Organ Fibrosis in a Humanized Graft versus Host Disease Murine Model

Allogeneic transplant rejection represents a medical complication that leads to high morbidity and mortality rates. There are no treatments to effectively prevent fibrosis; however, there is great interest in evaluating the use of perinatal mesenchymal stromal cells (MSCs) and other MSCs to prevent fibrosis associated with chronic rejection. In this study, we isolated human perinatal stromal cells (PSCs) from amnion (AM-PSC), placental villi (PV-PSC), and umbilical cord (UC-PSC) tissues, demonstrating the phenotypic characteristics of MSCs as well as a >70% expression of the immunomodulatory markers CD273 and CD210. The administration of a single dose (250,000 cells) of each type of PSC in a humanized graft versus host disease (hGvHD) NSG® murine model delayed the progression of the disease as displayed by weight loss and GvHD scores ranging at various levels without affecting the hCD3+ population. However, only PV-PSCs demonstrated an increased survival rate of 50% at the end of the study. Furthermore, a histopathological evaluation showed that only PV-PSC cells could reduce human CD45+ cell infiltration and the fibrosis of the lungs and liver. These findings indicate that not all PSCs have similar therapeutic potential, and that PV-PSC as a cell therapeutic may have an advantage for targeting fibrosis related to allograft rejection

Infusion of Some but Not All Types of Human Perinatal Stromal Cells Prevent Organ Fibrosis in a Humanized Graft versus Host Disease Murine Model

Allogeneic transplant rejection represents a medical complication that leads to high morbidity and mortality rates. There are no treatments to effectively prevent fibrosis; however, there is great interest in evaluating the use of perinatal mesenchymal stromal cells (MSCs) and other MSCs to prevent fibrosis associated with chronic rejection. In this study, we isolated human perinatal stromal cells (PSCs) from amnion (AM-PSC), placental villi (PV-PSC), and umbilical cord (UC-PSC) tissues, demonstrating the phenotypic characteristics of MSCs as well as a >70% expression of the immunomodulatory markers CD273 and CD210. The administration of a single dose (250,000 cells) of each type of PSC in a humanized graft versus host disease (hGvHD) NSG® murine model delayed the progression of the disease as displayed by weight loss and GvHD scores ranging at various levels without affecting the hCD3+ population. However, only PV-PSCs demonstrated an increased survival rate of 50% at the end of the study. Furthermore, a histopathological evaluation showed that only PV-PSC cells could reduce human CD45+ cell infiltration and the fibrosis of the lungs and liver. These findings indicate that not all PSCs have similar therapeutic potential, and that PV-PSC as a cell therapeutic may have an advantage for targeting fibrosis related to allograft rejection

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD <i>in vitro</i>. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment <i>in vitro</i> and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth <i>in vivo</i>. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.</p></div

Effect of EC13 on mammary gland morphology and uterine weight.

<p>A) Whole mounts of mammary glands of overiectomized C57BL/6 mice treated with E2, EC313 0.1 and 1.0 mg/kg. Magnification- upper panel-10X, lower panel-20X. B) Uterine weight of mice treated for 4 weeks with E2, EC313 0.1 and 1.0 mg/kg. Data represented are average uterine weight in milligrams ±SD (*P<0.001 vs E2-control). C-D) Total duct length and terminal end bud counts in the mammary gland whole mounts after 4 weeks of the treatment with test compounds (*P<0.001 vs E2-control).</p

Antiestrogenic effect of EC312/313 on cell viability and proliferation in vitro.

<p>A-B) Antiestrogenic effect of EC312 and EC313 on cell growth in the presence and absence of endogenous estrogens. T47D cells were plated at 30,000 cells per well in 5% FBS-RPMI and grown for 2 days before treatment. The cells were then incubated with EC312 and EC313 at indicated concentrations for 5 days and counted for cell number. *P<0.01 vs control. <b>C)</b> The compounds were treated as per the conditions above with and without E2 (0.1nM). Tested compounds were found to inhibit E2 induced cell proliferation as comparable to that of BZD (10nM). EC317 was used as a pure PR antagonist (*P<0.01 vs control).</p

EC312 and EC313 inhibiting DNA synthesis.

<p>A-B) Effect of EC312 and EC313 on cell proliferation as assessed by BrdU incorporation. T47D cells were plated in 96-well plates at a density of 10,000 cells per well. Two days later the culture medium (5% FCS-RPMI) was replaced with phenol red-free RPMI with 5% DCC-FCS. After 24 hours of starvation, the cells were treated with increasing doses of the compounds alone or in combination with E2 (0.1nM). The results are expressed as average OD value of quadruplicate wells (*P<0.01 vs control). C-D) Effect of EC312 and EC313 in inducing apoptosis in the presence or absence of endogenous estrogen in T47D cells. T47D cells were plated in 12 well plated at a density of 80,000 cells/well. Two days later culture media were replaced with phenol red-free RPMI containing 5% FBS or 5% DCC-FBS. After 24 hours of starvation, cells were treated with test compounds at increasing concentrations for 48 hours. Apoptosis was assessed by Caspase-Glo assay kit (Promega). The results were expressed as OD±SEM of 2 wells per treatment (*P<0.01 vs control).</p

Effect of EC313 on gene expression of proliferation, apoptotic markers and PR regulated genes.

<p>A-B), Gene expression in mammary glands of overiectomized C57BL/6 mice treated with E2, EC313 0.1 and 1.0 mg/kg. Columns are average of relative amount (ΔΔCt) of tested mRNA ± SD (n = 6) (**P<0.01 vs E2-control, *P<0.001 vs E2-control).</p

Glucocorticoid receptor (GR) antagonistic activity, Progesterone receptor (PR)—agonistic and antagonistic activity is evaluated and IC50 values as well as agonistic/antagonistic ratios were noted.

<p>ND- Not determined.</p

Effect of EC312 and EC313 on apoptosis, gene signatures of proliferation and protein expression levels.

<p>A) Effect of EC312 and EC313 (10nM) on apoptosis in the absence of endogenous estrogen and increasing concentrations of exogenous E2. We have selected the above concentration based on the fact that EC312 and EC313 abrogated E2 induced cell proliferation as comparable to that of BZD at 10nM). T47D cells were plated in 12 well plated at a density of 80,000 cells/well. Two days later culture media were replaced with phenol red-free RPMI containing 5% FBS or 5% DCC-FBS. After 24 hours of starvation, cells were treated with test compounds at increasing concentrations for 48 hours. Apoptosis was assessed by Caspase-Glo assay kit (Promega). The results were expressed as OD±SEM of 2 wells per treatment (*P<0.01 vs control). B) Effect of EC313 on gene signatures of proliferation. Gene expression of T47D cells in response to EC313 for anti-apoptotic genes. T47D cells were grown in phenol red-free RPMI medium containing 5% DCC-FBS for 24 hours and then treated with indicated doses of test compounds alone or in combination with E2 (0.1nM) (*P<0.01 vs E2-control). C) Effect of EC313 on gene signatures of proliferation. Gene expression of T47D cells in response to EC313 for pro-apoptotic genes. T47D cells were grown in phenol red-free RPMI medium containing 5% DCC-FBS for 24 hours and then treated with indicated doses of test compounds alone or in combination with E2 (0.1nM) (*P<0.01 vs E2-control). D) Effect of EC312 and BZD on the expression of ER, Cyclin D1 and phosphorylation of MAPK and AKT. T47D cells grown in 60-mm culture dishes in phenol red-free RPMI medium containing 5%DCC-FBS and treated with E2 (1nM) alone or in combination with EC312 or BZD (100nM) for 24 hours before preparation of cell lysate and analysis by western blot and quantification normalized by beta-actin.</p