Paola Pocar

Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications

Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10‐ to 14‐week‐old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus‐oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves.

Effect of Different Levels of Intracellular cAMP on the In Vitro Maturation of Cattle Oocytes and Their Subsequent Development Following In Vitro Fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus‐oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3‐isobutyl 1‐methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. 

Effects of Di(2-ethylhexyl) Phthalate (DEHP) on Female Fertility and Adipogenesis in C3H/N Mice

Background: Di(2-ethylhexyl) phthalate (DEHP) and its metabolites are known to affect lipid metabolism and adipogenesis, mainly by activation of peroxisome proliferator-activated receptors (PPARs). Exposure to DEHP has been linked with testicular impairment and male subfertility. However, the effects of DEHP on female reproductive health and metabolism have not been studied in detail. Objective: We examined the effects of dietary DEHP exposure on metabolism and fertility in female mice. Methods: In two independent approaches, female C3H/N mice were exposed to DEHP (0.05, 5, or 500 mg/kg of body weight per day) via their diet for 8 weeks, and we recorded food intake, weight gain, and litter size. After exposure, liver, visceral fat, and plasma from F0 females (study I) and F0 dams and their F1 offspring (study II) were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: In study I, DEHP-exposed F0 females (all dose groups) had a significant increase in body weight, food intake, and visceral adipose tissue compared with controls. In the 500-mg DEHP group, PPARα and PPARγ transcripts were significantly changed in liver tissue. In the same group, PPARγ mRNA was significantly reduced in liver but not in fat tissue. In addition, leptin and FABP4 (fatty acid binding protein 4) mRNA were increased in adipose tissue, whereas adiponectin was decreased. In study II, we detected a 100% abortion rate in F0 dams in the 500-mg group. F1 offspring exposed in utero and during lactation had an increase in visceral fat tissue and body weight. Conclusion: Fertility was impaired in mice exposed to high doses of DEHP, and body weight and visceral fat deposits were increased in mice exposed to environmentally relevant doses. Although F1 mice were exposed to DEHP only in utero and during lactation, we observed metabolic changes in the offspring of diet-exposed females.

Exposure to Di(2-ethyl-hexyl) phthalate (DEHP) in Utero and during Lactation Causes Long-Term Pituitary-Gonadal Axis Disruption in Male and Female Mouse Offspring

The present study examined the effects in mice of exposure to di(2-ethyl-hexyl) phthalate (DEHP) throughout pregnancy and lactation on the development and function of the pituitary-gonadal axis in male and female offspring once they have attained adulthood. Groups of two to three dams were exposed with the diet from gestational d 0.5 until the end of lactation, at 0, 0.05, 5, and 500 mg DEHP/kg · d. The experiment was repeated three times (total: seven to 10 dams per treatment). The 500-mg dose caused complete pregnancy failure, whereas exposure to doses of 0.05 and 5 mg did not affect pregnancy and litter size. In total, about 30 male and 30 female offspring per group were analyzed. Offspring of the DEHP-treated groups, compared with controls, at sexual maturity showed: 1) lower body weight (decrease 20-25%, P < 0.001); 2) altered gonad weight (testes were ∼13% lighter and ovaries ∼40% heavier; P < 0.001); 3) poor germ cell quality (semen was ∼50% less concentrated and 20% less viable, and ∼10% fewer oocytes reached MII stage, P < 0.001); 4) significant lower expression of steroidogenesis and gonadotropin-receptor genes in the gonads; and 5) up-regulated gonadotropin subunit gene expression in the pituitary. In conclusion, our findings suggest that, in maternally exposed male and female mice, DEHP acts on multiple pathways involved in maintaining steroid homeostasis. Specifically, in utero and lactational DEHP exposure may alter estrogen synthesis in both sexes. This, in turn, induces dysregulation of pituitary-gonadal feedback and alters the reproductive performance of exposed animals.

THE IN VITRO DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES CAN BE RELATED TO THE MORPHOLOGY OF THE OVARY

This study was designed to assess whether the developmental potential of bovine cumulus-oocyte complexes (COCs) could be related to the morphology of their originating ovary, providing a simple, noninvasive and objective selection criterion. Ovaries were divided into 3 categories on the basis of: A) presence of a follicle > 10 mm in diameter, B) presence of more than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm, and C) presence of less than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm. The COCs, isolated from ovaries of Category C, showed lower rates of maturation and blastocyst formation than those from Categories A and B. Moreover, blastocysts derived from Category C ovaries had fewer cells than those derived from the other 2 categories. It is concluded that ovarian morphology is a simple and noninvasive parameter for an effective selection of oocytes with better developmental competence.

Effects of pre-mating nutrition on mRNA levels of developmentally relevant genes in sheep oocytes and granulosa cells.

The present study was designed to investigate the relationship between pre-mating nutrition and the relative amounts of a panel of developmentally relevant genes in ovine oocytes and granulosa cells. Cast age ewes were fed a ration providing 0.5x (0.5 M) or 1.5x (1.5 M) live weight maintenance requirements for 2 weeks before slaughter. The ewes were synchronized and superovulated with FSH and pregnant mares serum gonadotropin. At slaughter, oocytes and granulosa cells were aspirated from follicles >2 mm in diameter and the relative abundance of 8 and 17 transcripts in oocytes and granulosa cells respectively were analyzed by semi-quantitative RT-PCR. In the oocytes, no differences between groups were observed for five transcripts (GDF9, BMP15, c-kit, glucose transporter 1 (SLC2A1), and hexokinase 1), but a lower amount of glucose transporter 3 (SLC2A3), sodium/glucose cotransporter 1 (SLC5A1), and Na(+)/K(+) ATPase mRNAs was detected in the 0.5 M group. Increased expression of PTGS2, HAS2, and the leptin receptor long form was observed in granulosa cells from the 0.5 M group. No differences between groups were observed for the other transcripts (early growth response factor-1, estrogen receptor-alpha, LH and FSH receptors, gremlin 1, pentraxin 3, KIT ligand, glucose transporters 1, 3, and 8, IGF1, IGF1 receptor, leptin receptor, and tumor necrosis factor-stimulated gene 6). Expression of leptin and sodium/glucose cotransporter 1 was not detected in both groups. The present data indicate that pre-mating nutrition is associated with alteration in the mRNA content in oocytes and surrounding follicle cells in ewes, which may account for the reduced reproductive performance typical of ewes that are fed a restricted ration for a short period of time before mating.

Cancer stem cells as targets for cancer therapy: selected cancers as examples

It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer-(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered.

In vitro reproductive toxicity of Polychlorinated biphenyls : Effects on oocyte maturation and developmental competence in cattle

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor‐1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus–oocytes complexes were exposed during IVM to serial concentrations of Aroclor‐1254 (between 1 μg/ml and 0.0001 μg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 μg/ml. Groups treated with 0.001 μg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures. Mol. Reprod. Dev. 58:411–416, 2001.

In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation, Energy and Oxidative Stress Parameters in a Large Animal Model

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.