San Antonio Nathan Shock Center: your one-stop shop for aging research.

With evolving cores, enrichment and training programs, and supported research projects, the San Antonio (SA) Nathan Shock Center has for 26 years provided critical support to investigators locally, nationally, and abroad. With its existing and growing intellectual capital, the SA Nathan Shock Center provides to local and external investigators an enhanced platform to conduct horizontally integrated (lifespan, healthspan, pathology, pharmacology) transformative research in the biology of aging, and serves as a springboard for advanced educational and training activities in aging research. The SA Nathan Shock Center consists of six cores: Administrative/Program Enrichment Core, Research Development Core, Aging Animal Models and Longevity Assessment Core, Pathology Core, Analytical Pharmacology and Drug Evaluation Core, and Integrated Physiology of Aging Core. The overarching goal of the SA Nathan Shock Center is to advance knowledge in the basic biology of aging and to identify molecular and cellular mechanisms that will facilitate the development of pharmacologic interventions and other strategies to extend healthy lifespan. In pursuit of this goal, we provide an innovative "one-stop shop" venue to accelerate transformative research in the biology of aging through our integrated research cores. Moreover, we aim to foster and promote career development of early-stage investigators in aging biology through our research development programs, to serve as a resource and partner to investigators from other Shock Centers, and to disseminate scientific knowledge and enhanced awareness about aging research. Overall, the SA Nathan Shock Center aims to be a leader in research that advances our understanding of the biology of aging and development of approaches to improve longevity and healthy aging.

Mobilization-based transplantation of young-donor hematopoietic stem cells extends lifespan in mice.

Mammalian aging is associated with reduced tissue regeneration and loss of physiological integrity. With age, stem cells diminish in their ability to regenerate adult tissues, likely contributing to age-related morbidity. Thus, we replaced aged hematopoietic stem cells (HSCs) with young-donor HSCs using a novel mobilization-enabled hematopoietic stem cell transplantation (HSCT) technology as an alternative to the highly toxic conditioning regimens used in conventional HSCT. Using this approach, we are the first to report an increase in median lifespan (12%) and a decrease in overall mortality hazard (HR: 0.42, CI: 0.273-0.638) in aged mice following transplantation of young-donor HSCs. The increase in longevity was accompanied by reductions of frailty measures and increases in food intake and body weight of aged recipients. Young-donor HSCs not only preserved youthful function within the aged bone marrow stroma, but also at least partially ameliorated dysfunctional hematopoietic phenotypes of aged recipients. This compelling evidence that mammalian health and lifespan can be extended through stem cell therapy adds a new category to the very limited list of successful anti-aging/life-extending interventions. Our findings have implications for further development of stem cell therapies for increasing health and lifespan.

Intranasal delivery of human umbilical cord Wharton's jelly mesenchymal stromal cells restores lung alveolarization and vascularization in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a devastating lung condition that develops in premature newborns exposed to prolonged mechanical ventilation and supplemental oxygen. Significant morbidity and mortality are associated with this costly disease and effective therapies are limited. Mesenchymal stem/stromal cells (MSCs) are multipotent cells that can repair injured tissue by secreting paracrine factors known to restore the function and integrity of injured lung epithelium and endothelium. Most preclinical studies showing therapeutic efficacy of MSCs for BPD are administered either intratracheally or intravenously. The purpose of this study was to examine the feasibility and effectiveness of human cord tissue-derived MSC administration given via the intranasal route. Human umbilical cord tissue MSCs were isolated, characterized, and given intranasally (500 000 cells per 20 µL) to a hyperoxia-induced rat model of BPD. Lung alveolarization, vascularization, and pulmonary vascular remodeling were restored in animals receiving MSC treatment. Gene and protein analysis suggest the beneficial effects of MSCs were attributed, in part, to a concerted effort targeting angiogenesis, immunomodulation, wound healing, and cell survival. These findings are clinically significant, as neonates who develop BPD have altered alveolar development, decreased pulmonary vascularization and chronic inflammation, all resulting in impaired tissue healing. Our study is the first to report the intranasal delivery of umbilical cord Wharton's jelly MSCs in experimental BPD is feasible, noninvasive, and an effective route that may bear clinical applicability.

Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach.

The directed differentiation of pluripotent stem cells to a desired lineage often involves complex and lengthy protocols. In order to study the requirements for differentiation in a systematic way, we present here methodology for an iterative approach using combinations of small molecules and biological factors. The factors are used in a cyclical process in which the best combination of factors and concentrations is selected in one round of testing, followed by a modification of the combination and subsequent rounds. While this may produce the desired differentiation in the cell population under study, it is also possible that other strategies may be needed to optimize the differentiation process. These strategies are described in this chapter.

Upcycling umbilical cords: bridging regenerative medicine with neonatology.

Preterm birth is a major health concern that affects 10% of all worldwide deliveries. Many preterm infants are discharged from the hospital with morbidities that lead to an increased risk for neurodevelopmental impairment, recurrent hospitalizations, and life-long conditions. Unfortunately, the treatment of these conditions is palliative rather than curative, which calls for novel and innovative strategies. Progress in regenerative medicine has offered therapeutic options for many of these conditions. Specifically, human umbilical cord mesenchymal stem cells (MSCs) and cord blood (UCB) cells have shown promise in treating adult-onset diseases. Unlike bone-marrow and embryonic derived stem cells, umbilical cord-derived cells are easily and humanely obtained, have low immunogenicity, and offer the potential of autologous therapy. While there are several studies to uphold the efficacy of umbilical cord MSCs in adult therapies, there remains an unmet need for the investigation of its use in treating neonates. The purpose of this review is to provide a summary of current information on the potential therapeutic benefits and clinical applicability of umbilical cord MSCs and UCB cells. Promising preclinical studies have now led to a research movement that is focusing on cell-based therapies for preterm infants.

Comparison of Preterm and Term Wharton's Jelly-Derived Mesenchymal Stem Cell Properties in Different Oxygen Tensions.

Mesenchymal stem cells (MSCs) have shown promise as therapeutic agents in treating morbidities associated with premature birth. MSCs derived from the human umbilical cord are easy to isolate and have low immunogenicity and a robust ability to secrete paracrine factors. To date, there are no studies evaluating preterm versus term umbilical cord tissue-derived MSCs. Therefore, our aim was twofold: (1) to compare stem cell properties in preterm versus term MSCs and (2) to examine the impact of oxygen tension on stem cell behavior. Umbilical cord tissue was obtained from 5 preterm and 5 term neonates. The cells were isolated and characterized as MSCs in accordance with the International Society for Cellular Therapy. We exposed MSCs to different oxygen tensions to examine the impact of environmental factors on cell performance. We studied the following stem cell properties: (i) motility, (ii) proliferation, (iii) senescence, (iv) cell viability, (v) colony-forming unit efficiency, and (vi) inflammatory cytokine expression. Under normoxia (21% O2), cells from preterm and term infants had similar properties. Under hypoxic conditions (1% O2), term MSCs had better cell proliferation; however, cells exposed to hyperoxia (90% O2) had the slowest motility and lowest cell viability (p < 0.05). There was no difference in the expression of senescence or cytokine expression between the groups. The term cells demonstrated more colony-forming efficiency than the preterm cells. In sum, our preliminary findings suggest that MSCs derived from term and preterm umbilical cords have similar characteristics, offering the potential of future autologous/allogeneic MSC transplants in neonates.

The Route by Which Intranasally Delivered Stem Cells Enter the Central Nervous System.

Intranasal administration is a promising route of delivery of stem cells to the central nervous system (CNS). Reports on this mode of stem cell delivery have not yet focused on the route across the cribriform plate by which cells move from the nasal cavity into the CNS. In the current experiments, human mesenchymal stem cells (MSCs) were isolated from Wharton's jelly of umbilical cords and were labeled with extremely bright quantum dots (QDs) in order to track the cells efficiently. At 2 h after intranasal delivery in immunodeficient mice, the labeled cells were found under the olfactory epithelium, crossing the cribriform plate adjacent to the fila olfactoria, and associated with the meninges of the olfactory bulb. At all times, the cells were separate from actual nerve tracts; this location is consistent with them being in the subarachnoid space (SAS) and its extensions through the cribriform plate into the nasal mucosa. In their location under the olfactory epithelium, they appear to be within an expansion of a potential space adjacent to the turbinate bone periosteum. Therefore, intranasally administered stem cells appear to cross the olfactory epithelium, enter a space adjacent to the periosteum of the turbinate bones, and then enter the SAS via its extensions adjacent to the fila olfactoria as they cross the cribriform plate. These observations should enhance understanding of the mode by which stem cells can reach the CNS from the nasal cavity and may guide future experiments on making intranasal delivery of stem cells efficient and reproducible.

Therapeutic potential of mesenchymal stromal cells for hypoxic ischemic encephalopathy: A systematic review and meta-analysis of preclinical studies.

INTRODUCTION: Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating neurologic condition with high mortality rates and long-term complications for surviving infants. Mesenchymal stem/stromal cells (MSCs) have emerged as novel therapeutic agents with promising results in experimental studies of HIE. The purpose of this study is to (a) methodically review the current preclinical literature describing MSC therapy in animal models of HIE, (b) quantify the effect size in regards to functional neurologic outcome, and (c) identify research gaps/limitations that should be addressed prior to future preclinical and clinical studies. METHODS: Adhering to the Systematic Review Protocol for Animal Intervention Studies, a systematic search of English articles was performed. Eligible studies were identified and data regarding study characteristics and outcome measures was extracted. After quality assessment, meta-analysis and meta-regression were performed to generate random effect size using standardized mean difference (SMD). Funnel plots and Egger's tests were utilized to evaluate for the presence of publication bias. RESULTS: A total of 19 studies met inclusion in the current systematic review. Meta-analysis revealed that MSCs have a significant positive effect on neurobehavioral outcome following HIE injury. Sensorimotor function was improved by 2.25 SMD (95% CI; 2.04-2.46) in cylinder rearing and 2.97 SMD (95% CI; 2.56-3.38) in rotarod. Likewise, cognitive function was improved by 2.76 SMD (95% CI; 2.53-2.98) on the water maze and 2.97 SMD (95% CI; 2.58-3.35) in object recognition. Stratification demonstrated an increased effect size depending on various study characteristics. CONCLUSIONS: Overall, these results suggest a promising role for MSCs in preclinical studies of HIE. MSC treatment demonstrates improved functional outcomes that are encouraging for future translational studies. While risk of bias and heterogeneity limited the strength of our meta-analysis, our results are consistent with those seen in this field of research.

Therapeutic potential of mesenchymal stem cells for diabetes.

Mesenchymal stem cells (MSCs) are self-renewing multipotent cells that have the capacity to secrete multiple biologic factors that can restore and repair injured tissues. Preclinical and clinical evidence have substantiated the therapeutic benefit of MSCs in various medical conditions. Currently, MSCs are the most commonly used cell-based therapy in clinical trials because of their regenerative effects, ease of isolation and low immunogenicity. Experimental and clinical studies have provided promising results using MSCs to treat diabetes. This review will summarize the role of MSCs on tissue repair, provide emerging strategies to improve MSC function and describe how these processes translate to clinical treatments for diabetes.

Overcoming barriers to reprogramming and differentiation in nonhuman primate induced pluripotent stem cells.

Induced pluripotent stem cells (iPS cells) generated by cellular reprogramming from nonhuman primates (NHPs) are of great significance for regenerative medicine and for comparative biology. Autologously derived stem cells would theoretically avoid any risk of rejection due to host-donor mismatch and may bypass the need for immune suppression post-transplant. In order for these possibilities to be realized, reprogramming methodologies that were initially developed mainly for human cells must be translated to NHPs. NHP studies have typically used pluripotent cells generated from young animals and thus risk overlooking complications that may arise from generating iPS cells from donors of other ages. When reprogramming is extended to a wide range of NHP species, available donors may be middle- or old-aged. Here we have pursued these questions by generating iPS cells from donors across the life span of the common marmoset (Callithrix jacchus) and then subjecting them to a directed neural differentiation protocol. The differentiation potential of different clonal cell lines was assessed using the quantitative polymerase chain reaction. The results show that cells derived from older donors often showed less neural marker induction. These deficits were rescued by a 24 h pretreatment of the cells with 0.5 % dimethyl sulfoxide. Another NHP that plays a key role in biological research is the chimpanzee (Pan troglodytes). iPS cells generated from the chimpanzee can be of great interest in comparative in vitro studies. We investigated if similar deficits in differentiation potential might arise in chimpanzee iPS cells reprogrammed using various technologies. The results show that, while some deficits were observed in iPS cell clones generated using three different technologies, there was no clear association with the vector used. These deficits in differentiation were also prevented by a 24 h pretreatment with 0.5 % dimethyl sulfoxide.

Endothelial cells regulate ß-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor.

Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of ß-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect ß-catenin activity in adrenocortical NCIH295R cells. Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of ß-catenin and an increase in ß-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced ß-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM. Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate ß-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.

Induced Pluripotent Stem Cells from Nonhuman Primates.

Induced pluripotent stem cells from nonhuman primates (NHPs) have unique roles in cell biology and regenerative medicine. Because of the relatedness of NHPs to humans, NHP iPS cells can serve as a source of differentiated derivatives that can be used to address important questions in the comparative biology of primates. Additionally, when used as a source of cells for regenerative medicine, NHP iPS cells serve an invaluable role in translational experiments in cell therapy. Reprogramming of NHP somatic cells requires the same conditions as previously established for human cells. However, throughout the process, a variety of modifications to the human cell protocols must be made to accommodate significant species differences.

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy.

Novel supercritical carbon dioxide impregnation technique for the production of amorphous solid drug dispersions: a comparison to hot melt extrusion.

The formulation of BCS Class II drugs as amorphous solid dispersions has been shown to provide advantages with respect to improving the aqueous solubility of these compounds. While hot melt extrusion (HME) and spray drying (SD) are among the most common methods for the production of amorphous solid dispersions (ASDs), the high temperatures often required for HME can restrict the processing of thermally labile drugs, while the use of toxic organic solvents during SD can impact on end-product toxicity. In this study, we investigated the potential of supercritical fluid impregnation (SFI) using carbon dioxide as an alternative process for ASD production of a model poorly water-soluble drug, indomethacin (INM). In doing so, we produced ASDs without the use of organic solvents and at temperatures considerably lower than those required for HME. Previous studies have concentrated on the characterization of ASDs produced using HME or SFI but have not considered both processes together. Dispersions were manufactured using two different polymers, Soluplus and polyvinylpyrrolidone K15 using both SFI and HME and characterized for drug morphology, homogeneity, presence of drug-polymer interactions, glass transition temperature, amorphous stability of the drug within the formulation, and nonsink drug release to measure the ability of each formulation to create a supersaturated drug solution. Fully amorphous dispersions were successfully produced at 50% w/w drug loading using HME and 30% w/w drug loading using SFI. For both polymers, formulations containing 50% w/w INM, manufactured via SFI, contained the drug in the γ-crystalline form. Interestingly, there were lower levels of crystallinity in PVP dispersions relative to SOL. FTIR was used to probe for the presence of drug-polymer interactions within both polymer systems. For PVP systems, the nature of these interactions depended upon processing method; however, for Soluplus formulations this was not the case. The area under the dissolution curve (AUC) was used as a measure of the time during which a supersaturated concentration could be maintained, and for all systems, SFI formulations performed better than similar HME formulations.

Long-lived species have improved proteostasis compared to phylogenetically-related shorter-lived species.

Our previous studies have shown that the liver from Naked Mole Rats (NMRs), a long-lived rodent, has increased proteasome activity and lower levels of protein ubiquitination compared to mice. This suggests that protein quality control might play a role in assuring species longevity. To determine whether enhanced proteostasis is a common mechanism in the evolution of other long-lived species, here we evaluated the major players in protein quality control including autophagy, proteasome activity, and heat shock proteins (HSPs), using skin fibroblasts from three phylogenetically-distinct pairs of short- and long-lived mammals: rodents, marsupials, and bats. Our results indicate that in all cases, macroautophagy was significantly enhanced in the longer-lived species, both at basal level and after induction by serum starvation. Similarly, basal levels of most HSPs were elevated in all the longer-lived species. Proteasome activity was found to be increased in the long-lived rodent and marsupial but not in bats. These observations suggest that long-lived species may have superior mechanisms to ensure protein quality, and support the idea that protein homeostasis might play an important role in promoting longevity.

Hedgehog-signaling is upregulated in non-producing human adrenal adenomas and antagonism of hedgehog-signaling inhibits proliferation of NCI-H295R cells and an immortalized primary human adrenal cell line.

Hedgehog (Hh)-signaling pathway is important in embryonic development. Activation of Hh-signaling is associated with tumorigenesis. Recent studies demonstrate that Hh-signaling is involved in the development of the adrenal gland in mice and is important in regulating adrenal proliferation. We studied the expression of Sonic hedgehog (SHH), Smoothened (SMO), Patched1 (PTCH1) and GLI family zinc finger 1 (GLI1) in human adrenal and in adrenocortical tumors using immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Modulation of GLI1 and SMO messenger ribonucleic acid (mRNA) expression was investigated with forskolin. The role of Hh-signaling was studied in NCI-H295R cells and in an immortalized primary cell line using the Hh-agonist smoothened agonist (SAG) and the Hh-antagonist cyclopamine. The Hh-pathway components SHH, GLI1, PTCH1 and SMO were detectable in all adrenal glands. While in cortisol-producing adenomas (CPA), Hh-signaling expression levels were comparable to that in normal adrenal cortex, a much higher mRNA expression of GLI1, SMO and SHH was observed in non-producing adenomas (NPA). Interestingly, stimulation of cultured adrenal cells with forskolin led to a decrease in expression of GLI1 and SMO mRNAs. Antagonism of Hh-signaling resulted in a lower proliferation rate of adrenocortical cells, while Hh-agonism had no significant effect on adrenal cell proliferation. Our data show Hh-signaling activity in adult adrenal glands. Activation of the PKA pathway results in lower expression of Hh-signaling proteins. This might explain the lower expression of the Hh components GLI1 and SMO in CPA in comparison to the higher expression in NPA. Hh-signaling might be involved in the tumorigenesis of NPA.

Derivation of induced pluripotent stem cells from the baboon: a nonhuman primate model for preclinical testing of stem cell therapies.

Development of effective pluripotent stem cell-based therapies will require safety and efficacy testing in a clinically relevant preclinical model such as nonhuman primates (NHPs). Baboons and macaques are equally similar to humans genetically and both have been extensively used for biomedical research. Macaques are preferred for human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) research whereas baboons are preferred for transplantation studies because of the greater similarity of their anatomy and immunogenetic system to those of humans. We generated four induced pluripotent stem cell (iPSC) lines from skin cells of the olive baboon (Papio anubis). Each line shows the distinct morphology of primate pluripotent stem cells, including flat colonies with well-defined borders and a high nuclear/cytoplasm ratio. Each is positive for the pluripotency markers OCT4, SOX2, NANOG, and SSEA4. Pluripotency was confirmed in two lines by teratoma formation with representative tissues from each germ layer, whereas a third produced cells from all three germ layers following embryoid body differentiation. Three lines have a normal male karyotype and the fourth is missing the short arm of one copy of chromosome 18. This may serve as an in vitro model for the human developmental disorder 18p-, which impacts 1 in 50,000 births/year. These iPSC lines represent the first step toward establishing the baboon as a NHP model for developing stem cell-based therapies.

Longitudinal analysis of short-term high-fat diet on endothelial senescence in baboons.

A high-fat diet is a major risk factor for atherosclerosis. We conducted a longitudinal investigation to determine whether vascular endothelial senescence is involved in the mechanism by which a high-fat diet promotes atherogenesis. We challenged 10 baboons (Papio sp.) with a high-cholesterol high-fat (HCHF) diet for 7 weeks. In addition to multiple changes in plasma lipid profiles, inflammatory status, and endothelial functions in each individual, we found that levels of total serum cholesterol (TSC) and monocyte chemotactic protein-1 (MCP-1) were negatively and significantly correlated with endothelial nitric oxide synthase (eNOS) levels in endothelial cells while the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were significantly correlated with nitric oxide levels in plasma within this time window. Most important, we observed that senescence-associated ß-galactosidase (SA-ß-gal) activities in endothelial cells harvested at 7 weeks after initiation of HCHF diet were significantly elevated by comparison with cells isolated from the same animals prior to dietary challenge. The SA-ß-gal activities correlated significantly with the elevations of TSC, LDL-cholesterol, HDL-cholesterol, and IL-8 after 7 weeks of HCHF diet and with the changes of TSC and TNF-α levels after 3 weeks of HCHF diet. Our data indicate that the HCHF diet caused hyperlipidemia and prominent inflammation, which subsequently will cause endothelial dysfunction and promote senescence. The present study is the first to demonstrate the sequential and interactive changes as a consequence of an HCHF dietary challenge and establish a potential mechanism underlying the etiology of diet-induced atherogenesis in a nonhuman primate.

Directed neural differentiation of induced pluripotent stem cells from non-human primates.

Induced pluripotent stem cells (iPS cells) are important for the future development of regenerative medicine involving autologous cell therapy. Before autologous cell therapy can be applied to human patients, suitable animal models must be developed, and in this context non-human primate models are critical. We previously characterized several lines of marmoset iPS cells derived from newborn skin fibroblasts. In the present studies, we explored methods for the directed differentiation of marmoset iPS cells in the neuroectodermal lineage. In this process we used an iterative process in which combinations of small molecules and protein factors were tested for their effects on mRNA levels of genes that are markers for the neuroectodermal lineage. This iterative process identified combinations of chemicals/factors that substantially improved the degree of marker gene expression over the initially tested combinations. This approach should be generally valuable in the directed differentiation of pluripotent cells for experimental cell therapy.

WNT4 acts downstream of BMP2 and functions via ß-catenin signaling pathway to regulate human endometrial stromal cell differentiation.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFß superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active ß-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/ß-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of ß-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/ß-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.