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.

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.

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.

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.

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.

Multimodal bioimaging using rare earth doped Gd2O2S: Yb/Er phosphor with upconversion luminescence and magnetic resonance properties.

While infrared upconversion imaging using halide nanoparticles are so common the search for a very efficient halide free upconverting phosphors is still lacking. In this article we report Gd2O2S:Yb/Er,YbHo,YbTm systems as a very efficient alternative phosphors that show upconversion efficiency comparable or even higher than existing halide phosphors. While the majority of rare earth dopants provide the necessary features for optical imaging, the paramagnetic Gd ion also contributes to the magnetic imaging,thereby resulting in a system with bimodal imaging features. Results from imaging of the nanoparticles together with aggregates of cultured cells have suggested that imaging of the particles in living animals may be possible. In vitro tests revealed no signficant toxicity because no cell death was observed when the nanoparticles were in the presence of growing cells in culture. Measurement of the magnetization of the phosphor shows that the particles are strongly magnetic, thus making them suitable as an MRI agent.

Patient-specific induced pluripotent stem cells in neurological disease modeling: the importance of nonhuman primate models.

The development of the technology for derivation of induced pluripotent stem (iPS) cells from human patients and animal models has opened up new pathways to the better understanding of many human diseases, and has created new opportunities for therapeutic approaches. Here, we consider one important neurological disease, Parkinson's, the development of relevant neural cell lines for studying this disease, and the animal models that are available for testing the survival and function of the cells, following transplantation into the central nervous system. Rapid progress has been made recently in the application of protocols for neuroectoderm differentiation and neural patterning of pluripotent stem cells. These developments have resulted in the ability to produce large numbers of dopaminergic neurons with midbrain characteristics for further study. These cells have been shown to be functional in both rodent and nonhuman primate (NHP) models of Parkinson's disease. Patient-specific iPS cells and derived dopaminergic neurons have been developed, in particular from patients with genetic causes of Parkinson's disease. For complete modeling of the disease, it is proposed that the introduction of genetic changes into NHP iPS cells, followed by studying the phenotype of the genetic change in cells transplanted into the NHP as host animal, will yield new insights into disease processes not possible with rodent models alone.

Nonhuman primate induced pluripotent stem cells in regenerative medicine.

Among the various species from which induced pluripotent stem cells have been derived, nonhuman primates (NHPs) have a unique role as preclinical models. Their relatedness to humans and similar physiology, including central nervous system, make them ideal for translational studies. We review here the progress made in deriving and characterizing iPS cell lines from different NHP species. We focus on iPS cell lines from the marmoset, a small NHP in which several human disease states can be modeled. The marmoset can serve as a model for the implementation of patient-specific autologous cell therapy in regenerative medicine.

Adrenarche: a cell biological perspective.

Adrenarche is a cell biological and endocrinological puzzle. The differentiation of the zona reticularis in childhood in humans requires special techniques for study because it is confined to humans and possibly a small number of other primates. Despite the rapid progress in the definition of adrenocortical stem/progenitor cells in the mouse, the factors that cause the differentiation of adrenocortical cells into zonal cell types have not been identified. There are, however, many candidates in the Wnt, Hedgehog, and other families of signaling molecules. A suitable system for identifying authentic stem cells, capable of differentiation into all zones, has yet to be developed. It is proposed here that the in vitro differentiation of pluripotent cells, combined with appropriate in vitro and in vivo methods for validating authentic adrenocortical stem cells, is a promising approach to solving these questions.

Stress resistance in the naked mole-rat: the bare essentials - a mini-review.

BACKGROUND: Studies comparing similar-sized species with disparate longevity may elucidate novel mechanisms that abrogate aging and prolong good health. We focus on the longest living rodent, the naked mole-rat. This mouse-sized mammal lives ~8 times longer than do mice and, despite high levels of oxidative damage evident at a young age, it is not only very resistant to spontaneous neoplasia but also shows minimal decline in age-associated physiological traits. OBJECTIVES: We assess the current status of stress resistance and longevity, focusing in particular on the molecular and cellular responses to cytotoxins and other stressors between the short-lived laboratory mouse and the naked mole-rat. RESULTS: Like other experimental animal models of lifespan extension, naked mole-rat fibroblasts are extremely tolerant of a broad spectrum of cytotoxins including heat, heavy metals, DNA-damaging agents and xenobiotics, showing LD(50) values between 2- and 20-fold greater than those of fibroblasts of shorter-lived mice. Our new data reveal that naked mole-rat fibroblasts stop proliferating even at low doses of toxin whereas those mouse fibroblasts that survive treatment rapidly re-enter the cell cycle and may proliferate with DNA damage. Naked mole-rat fibroblasts also show significantly higher constitutive levels of both p53 and Nrf2 protein levels and activity, and this increases even further in response to toxins. CONCLUSION: Enhanced cell signaling via p53 and Nrf2 protects cells against proliferating with damage, augments clearance of damaged proteins and organelles and facilitates the maintenance of both genomic and protein integrity. These pathways collectively regulate a myriad of mechanisms which may contribute to the attenuated aging profile and sustained healthspan of the naked mole-rat. Understanding how these are regulated may be also integral to sustaining positive human healthspan well into old age and may elucidate novel therapeutics for delaying the onset and progression of physiological declines that characterize the aging process.

Biosensor technology in aging research and age-related diseases.

Cell- and tissue-based biosensors comprise genetically engineered proteins that are incorporated into cells ex vivo or into cells of tissues in vivo. They enable the investigator to sense levels of hormones, drugs, or toxins, continuously and noninvasively, using biophotonics or other physical principles, and could potentially be used over the entire lifespan of an experimental animal. The present work reviews the state of the art of cell- and tissue-based biosensors and discusses how they could be of value in aging research. Examples of recently developed biosensors are given, including those that detect levels of a cytokine (TNFα) and drugs (activators of the mTOR pathway). Finally, we discuss the hurdles that would have to be overcome for biosensor technology to be used in humans in monitoring health status and disease treatment in late life.