Human Chorionic Gonadotropin Improves the Proliferation and Regenerative Potential of Bone Marrow Adherent Stem Cells and the Immune Tolerance of Fetal Microchimeric Stem Cells In Vitro.

Nongonadal tissues express luteinizing hormone-chorionic gonadotropin receptors (LHCG-R) which are essential for their growth during fetal development. Adult mesenchymal stem/stromal cells (MSCs) have been shown to express functional LHCG-R outside pregnancy conditions, making them susceptible to hCG stimulation. In the present study we tested the effect of hCG treatment on bone marrow (BM) derived adherent stem cells in vitro, isolated from a parous women, mother of male sons, in order to evaluate its effect on maternal MSCs and in the same time on fetal microchimeric stem cells (FMSCs), to better understand the outcomes of this safe and affordable treatment on cell proliferation and expression of pluripotency genes. Our study highlights the beneficial effects of hCG exposure on gene regulation in bone marrow adherent stem cells through the upregulation of pluripotency genes and selection of more primitive mesenchymal stem cells with a better differentiation potential. Validation of these effects on MSCs and FMSCs long after parturition in vivo represents a close perspective as it could set the premises of a new mobilization strategy for the stem cell transplantation procedures in the clinical setting.

The effect of lipopolysaccharide on the expression level of immunomodulatory and immunostimulatory factors of human amniotic epithelial cells.

OBJECTIVE: Human amniotic epithelial cells (hAECs) are a novel source of stem cells and have immunomodulatory effects on both the innate and adoptive immune system. hAECs can differentiate into multiple cell lineages that make them a suitable cell source for regenerative medicine. These cells express multiple toll-like receptors (TLRs) and respond to various TLR ligands. This study aimed to evaluate the effect of lipopolysaccharide (LPS), a TLR4 ligand, on the level of immunomodulatory and immunostimulatory factors of hAECs. RESULTS: Our results indicated that LPS had the ability to up-regulate the expression of prostaglandin E2 synthase and transforming growth factor-beta1 in hAECs. However, there was no change in the level of interleukin-1beta, interleukin-6 and interleukin-10 in hAECs when were stimulated with LPS. In addition, we observed tumor necrosis factor-alpha was only expressed at very low level in some of hAECs samples which its expression was independent of the effects of LPS.

Epithelial cell adhesion molecule fragments and signaling in primary human liver cells.

Epithelial Cell Adhesion Molecule (EpCAM), or CD326, is a trans-membrane glycoprotein expressed by multiple normal epithelia as well as carcinoma. Human hepatic stem cells and bile duct epithelium of the liver are EpCAM positive. In tumor cell lines, its intracellular domain can be released after cleavage of the extracellular domain. Within the cell nucleus, it induces cell proliferation, but cleavage depends on cell contact. Fragments of various lengths have been described in tumor cells. Despite its described important role in proliferation in tumor cells, there is not much known about the expression and role of EpCAM fragments in primary human liver cells. Here, we demonstrate that EpCAM protein fragments and function are considerable different between tumor cells, normal fetal and adult liver cells. Contrary to previously reported findings in tumor cells, gene knockdown or treatment with an inhibitor of the cleavage enzyme ADAM17 (TACE) rather increased cell numbers in primary human fetal liver-derived EpCAM-positive cells. EpCAM fragment sizes were not affected by treatment with inhibitor. Knockdown of EPCAM gene expression by siRNA in sorted cells did not significantly affect proliferation-associated genes or cell numbers. The intracellular domain could not be detected within cell nuclei of fetal and adult liver cells. In conclusion, signaling through the intracellular domain of EpCAM appears to be a mechanism that induces proliferation specifically in tumorigenic cells but not in normal primary EpCAM-positive liver cells.

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy.

Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model.

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc.

Prenatal acetaminophen induces liver toxicity in dams, reduces fetal liver stem cells, and increases airway inflammation in adult offspring.

BACKGROUND & AIMS: During pregnancy, acetaminophen is one of the very few medications recommended by physicians to treat fever or pain. Recent insights from epidemiological studies suggest an association between prenatal acetaminophen medication and an increased risk for development of asthma in children later in life. The underlying pathogenesis of such association is still unknown. METHODS: We aimed to develop a mouse model to provide insights into the effect of prenatal acetaminophen on maternal, fetal and adult offspring's health. The toxic effect of acetaminophen was studied in mice on 1) maternal liver; mirrored by biomarkers of liver injury, centrilobular necrosis, and infiltration of granulocytes; 2) fetal liver; reflected by the frequency of hematopoietic stem cells, and 3) postnatal health; evaluated by the severity of allergic airway inflammation among offspring. RESULTS: We observed an increased susceptibility towards acetaminophen-induced liver damage in pregnant mice compared to virgins. Moreover, hematopoietic stem cell frequency in fetal liver declined in response to acetaminophen. Furthermore, a greater severity of airway inflammation was observed in offspring of dams upon prenatal acetaminophen treatment, identified lung infiltration by leukocytes and eosinophil infiltration into the airways. CONCLUSION: Our newly developed mouse model on prenatal use of acetaminophen reflects findings from epidemiological studies in humans. The availability of this model will allow improvement in our understanding of how acetaminophen-related hepatotoxicity is operational in pregnant individuals and how an increased risk for allergic diseases in response to prenatal acetaminophen is mediated. Such insights, once available, may change the recommendations for prenatal acetaminophen use.

The LINA cohort: Cord blood eosinophil/basophil progenitors predict respiratory outcomes in early infancy.

RATIONALE: Cord blood eosinophil/basophil progenitor cells (Eo/B) of high risk infants have been shown to predict respiratory illnesses in infancy. Here we investigated this association in a population-based cohort. Furthermore, we analysed whether newborns Th1/Th2 balance and prenatal environmental exposure impact Eo/B recruitment. METHODS: In a sub-cohort of the LINA study cord blood mononuclear cells were used for methylcellulose assays to assess Eo/B differentiation. Questionnaires were recorded during pregnancy and annually thereafter. Volatile organic compounds were measured during pregnancy and cord blood cytokines after ex vivo stimulation. RESULTS: Cord blood IL-4 and IL-13 positively correlated with Eo/B. Tobacco smoke related benzene was also positively associated with Eo/B. Enhanced Eo/B numbers increased the risk for wheezing within the first 24 months. CONCLUSIONS: The association between cord blood Eo/B and respiratory illnesses is not restricted to high-risk children. Prenatal environmental exposure and a Th2 milieu at birth contribute to Eo/B recruitment.

Involvement of extracellular signal-regulated kinase (ERK1/2)-p53-p21 axis in mediating neural stem/progenitor cell cycle arrest in co-morbid HIV-drug abuse exposure.

Neurological complications in opioid abusing Human Immunodeficiency Virus-1 (HIV-1) patients suggest enhanced neurodegeneration as compared to non-drug abusing HIV-1 infected population. Neural precursor cells (NPCs), the multipotent cells of the mammalian brain, are susceptible to HIV-1 infection and as opiates also perturb their growth kinetics, detailed mechanistic studies for their co-morbid exposure are highly warranted. Using a well characterized in vitro model of human fetal brain-derived neural precursor cells, we investigated alterations in NPC properties at both acute and chronic durations. Chronic morphine and Tat treatment attenuated proliferation in NPCs, with cells stalled at G1-phase of the cell cycle. Furthermore HIV-Tat and morphine exposure increased activation of extracellular signal-regulated kinase-1/2 (ERK1/2), enhanced levels of p53 and p21, and decreased cyclin D1 and Akt levels in NPCs. Regulated by ERK1/2 and p53, p21 was found to be indispensible for Tat and morphine mediated cell cycle arrest. Our study elaborates on the cellular and molecular machinery in NPCs and provides significant mechanistic details into HIV-drug abuse co-morbidity that may have far reaching clinical consequences both in pediatric as well as adult neuroAIDS.

Opposing actions of ethanol and nicotine on microRNAs are mediated by nicotinic acetylcholine receptors in fetal cerebral cortical-derived neural progenitor cells.

BACKGROUND: Ethanol (EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells (NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific microRNAs (miRNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (miR-9, miR-21, miR-153, and miR-335), a nicotine-sensitive miRNA (miR-140-3p), and mRNAs for nicotinic acetylcholine receptor (nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent. METHODS: Gestational day 12.5 mouse fetal murine cerebral cortical-derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of miRNAs, miRNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: EtOH suppressed the expression of known EtOH-sensitive miRNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these miRNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and ß2 nAChR transcripts. CONCLUSIONS: EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive miRNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts miRNA regulatory networks that are important for NSC maturation.

Stem cell niches exposed to tobacco smoke.

Stem cells (SC) were identified in both fetal and adult tissues. They appear a great potential for proliferation and differentiation. SC functions are affected by their local microenvironment, known as a SC niche, composed of extracellular matrix (ECM) and neighboring cells of other types. Both fetal and adult SC niches are potential targets for environmental pollutions. Environmental Tobacco Smoke (ETS) is one of the most dangerous sources of toxic and carcinogenic compounds. Adverse effect of tobacco smoke components on SC includes both direct influence on the cells and their regulatory mechanisms, as well as changes in SC microenvironment. Both mechanisms contribute to limitation of regenerative potential of the tissues. As targets for tobacco smoke during long-lasting active or passive smoking, SC can initiate malignant transformation. So called, cancer stem cells (CSC) exhibit some similarities as compared with SC, for example abilities of self-renewal and asymmetric divisions. Cancer treatment focused on CSC may contribute to increased efficiency of standard therapeutic methods. To sum up, effects of stem cells exposure to tobacco smoke may be particularly important in the context of tissue regeneration and carcinogenesis. Furthermore, SC exposure to tobacco smoke components may result in reduced number and quality of stem cells deposited in the tissue reservoirs. As a result the quality of SC derived from fetal (cord blood, amniotic fluid, placenta) and adult donors (i.e. bone marrow) for transplantations might be much reduced.

BDNF promotes EGF-induced proliferation and migration of human fetal neural stem/progenitor cells via the PI3K/Akt pathway.

Neurogenesis is a complex process, which contributes to the ability of the adult brain to function normally and adapt to diseases. Epidermal growth factor (EGF) is known to play an important role in neurogenesis; however, the underlying mechanism is still unclear. Here, we hypothesized that brain-derived neurotrophic factor (BDNF) can enhance the effect of EGF on neurogenesis. Using in vitro cell culture of aborted human fetal brain tissues, we investigated proliferation and migration of neural stem/progenitor cells (NSPCs) after treatment with EGF and different concentrations of BDNF. EGF stimulated proliferation and migration of NSPCs, and this effect was significantly enhanced by co-incubation with BDNF. In the NSPCs treated with 50 ng/mL BDNF, BrdU incorporation was significantly increased (from 7.91% to 17.07%), as compared with that in the control. Moreover, the number of migrating cells was at least 2-fold higher than that in the control. Furthermore, phosphorylation of Akt-1 was increased by BDNF treatment, as well. By contrast, the enhancing effect of BDNF on EGF-induced proliferation and migration of NSPCs were abolished by an inhibitor of PI3K, LY294002. These findings suggest that BDNF promotes EGF-induced proliferation and migration of NSPC through the PI3K/Akt pathway, providing significant insights into not only the mechanism underlying EGF-induced neurogenesis but also potential neuronal replacement strategies to treat brain damage.

Creation of an in vitro microenvironment to enhance human fetal synovium-derived stem cell chondrogenesis.

Our aim was to assess the feasibility of the sequential application of extracellular matrix (ECM) and low oxygen to enhance chondrogenesis in human fetal synovium-derived stem cells (hfSDSCs). Human fetal synovial fibroblasts (hfSFs) were characterized and found to include hfSDSCs, as evidenced by their multi-differentiation capacity and the surface phenotype markers typical of mesenchymal stem cells. Passage-7 hfSFs were plated on either conventional plastic flasks (P) or ECM deposited by hfSFs (E) for one passage. Passage-8 hfSFs were then reseeded for an additional passage on either P or E. The pellets from expanded hfSFs were incubated in a serum-free chondrogenic medium supplemented with 10 ng/ml transforming growth factor-ß3 under either normoxia (21% O(2); 21) or hypoxia (5% O(2); 5) for 14 days. Pellets were collected for evaluation of the treatments (EE21, EE5, EP21, EP5, PE21, PE5, PP21, and PP5) on expanded hfSF chondrogenesis by using histology, immunostaining, biochemistry, and real-time polymerase chain reaction. Our data suggest that, compared with seeding on conventional plastic flasks, hfSFs expanded on ECM exhibit a lower expression of senescence-associated ß-galactosidase and an enhanced level of stage-specific embryonic antigen-4. ECM-expanded hfSFs also show increased cell numbers and an enhanced chondrogenic potential. Low oxygen (5% O(2)) during pellet culture enhances hfSF chondrogenesis. Thus, we demonstrate, for the first time, the presence of stem cells in hfSFs, and that modulation of the in vitro microenvironment can enhance hfSDSC chondrogenesis. hfSDSCs might represent a promising cell source for cartilage tissue engineering and regeneration.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering.

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

Etoposide induces G2/M arrest and apoptosis in neural progenitor cells via DNA damage and an ATM/p53-related pathway.

Etoposide (VP-16), an anti-tumor agent, is a topoisomerase II inhibitor that causes DNA damage. In our previous studies, it was shown that VP-16 induces S-phase accumulation and G2/M arrest, eventually resulting in apoptosis, through p53-related pathway in the mouse fetal brain. We injected 4 mg/kg of VP-16 into pregnant mice on day 12 of gestation, and the fetuses were investigated for the cell cycle checkpoint and mechanism of apoptosis. The transition of the neural progenitor cells in the fetuses was delayed as compared to that in the control, and most of the apoptotic cells were BrdU positive. VP-16-induced S-phase accumulation was brought about by the acceleration of G1/S transition rather than by the inhibition of S-phase progression. Phosphorylation of ataxia telangiectasia-mutated kinase (ATM) at Ser1981 and gammaH2AX after VP-16 treatment showed DNA damage. p53 was phosphorylated at Ser15 and 20 and increased after activation of the ATM kinase pathway. Cdc25A degradation might induce the inhibition of S-phase progression. It is supposed that an increase in cyclin A might accelerate G1/S progression. It is also indicated that VP-16-induced G2/M arrest is caused by p21, which inactivates cyclin B-Cdc2 complex and eventually prevents mitotic entry. In p53-deficient fetal brains, G2/M and apoptosis were almost abrogated, although S-phase accumulation still occurred. It is suggested that VP-16 induced p53-independent S-phase accumulation, and p53-dependent G2/M arrest and apoptosis of the neural progenitor cells in fetal mouse brain.

In utero exposure to benzene disrupts fetal hematopoietic progenitor cell growth via reactive oxygen species.

It is hypothesized that the increasing incidence of childhood leukemia may be due to in utero exposure to environmental pollutants, such as benzene, but the mechanisms involved remain unknown. We hypothesize that reactive oxygen species (ROS) contribute to the deregulation of fetal hematopoiesis caused by in utero benzene exposure. To evaluate this hypothesis, pregnant C57Bl/6N mice were exposed to benzene or polyethylene glycol-conjugated catalase (PEG-catalase) (antioxidative enzyme) and benzene. Colony formation assays on fetal liver cells were performed to measure erythroid and myeloid progenitor cell growth potential. The presence of ROS in CD117(+) fetal liver cells was measured by flow cytometric analysis. Oxidative cellular damage was assessed by Western blot analysis of 4-hydroxynonenol (4-HNE) and nitrotyrosine products, as well as reduced to oxidized glutathione ratios. Alterations in the redox-sensitive signaling pathway nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-kappaB) were measured by Western blot analysis of Inhibitor of NF-kB-alpha (IkappaB-alpha) protein levels in fetal liver tissue. In utero exposure to benzene caused a significant increase in ROS production and significantly altered fetal liver erythroid and myeloid colony numbers but did not increase the levels of 4-HNE or nitrotyrosine products or alter reduced to oxidized glutathione ratios. However, in utero exposure to benzene did cause a significant decrease in fetal liver IkappaB-alpha protein levels, suggesting activation of the NF-kappaB pathway. Benzene-induced ROS formation, abnormal colony growth, and decreased IkappaB-alpha levels were all abrogated by pretreatment with PEG-catalase. These results suggest that ROS play a key role in the development of in utero-initiated benzene toxicity potentially through disruption of hematopoietic cell signaling pathways.

Limited Ca2+ and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells.

The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to cAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by cAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs' differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity.

Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells.

The aim of this study is to investigate effects of 5-azacytidine (5-aza) induction duration on differentiation of bone marrow mesenchymal stem cells (MSCs) from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. hfMSCs were stimulated with 10 micromol/l 5-aza for 24 h (group A), 48 h (group B) and 21 days (group C), respectively. During the induction, 30-40% of the cells gradually enlarged, elongated, connected with adjoining cells and formed myotube-like structures, branches and string-bead-like nuclei. Some of the cells congregated into cell clusters or strips. After the induction, numerous myofilaments in the cytoplasm and conjunction of intercalated disc-like structure between adjoining cells were observed. The induced cells expressed messenger ribonucleic acids (mRNAs) and proteins of myocardium-specific alpha-actin, sarcomeric beta-myocin heavy chain and troponin-T. The positive cell percentages for the three antigens in group C were each significantly higher than those antigens in group A and B (P<0.01) and the cell population doubling time (PDT) of group C was longer than those of group A and B (P<0.01). These indicate that 21-d induction with 10 micromol/l 5-aza slows down proliferation speed of hfMSCs but increases differentiation rate of hfMSCs into cardiomyocyte-like cells if compared with 24-48 h induction.

Toxicity of Trigonella foenum graecum (Fenugreek) in bone marrow cell proliferation in rat.

Fenugreek has a wide range of medical applications and its medicinal use has been clear in several studies, however, few studies are available on effects on haematopoietic stem cell of bone marrow. The goal of the present study was to investigate the effect of Fenugreek on fetal macroscopic diameters and microscopic bone marrow cell histological changes in its teratogenic dosages. Fenugreek decoction was dissolved in 1.5 milliliter distilled water and injected intraperitoneumly in three dosages of 0.8 g/kg, 1.6 g/kg, and 3.2 g/kg for three groups of Wistar female rats mated by Wistar male. For another group (as control group) only 1.5 milliliter distilled water was injected. Bone marrow tissue was prepared from rat fetus and was cut using a microtome and stained with hematoxylin and eosin. Sections were evaluated for changes using light microscope. LD(50) for the measurement of teratogenic dosage of fenugreek was 4.1 and 3.5 g/kg in female and male rat, respectively. There was a positive relation between the injected drug dosage and fetal mortality rate. Among all fetal diameters, ear to ear diameter was decreased in groups received Fenugreek decoction. The severity of stem cell histological changes caused by 3.2 g/kg drug injection was lower than distilled water injection and in evaluation of other cells, differences in the severity of histological changes across three groups with different drug dosages and control group was detected. Fenugreek in teratogenic dosages can decrease the severity of bone marrow cell proliferation and increase fetal mortality rate.

Release of proinflammatory mediators and expression of proinflammatory adhesion molecules by endothelial progenitor cells.

Cell therapy with endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote angiogenesis or endothelial repair. Although the release of angiogenic paracrine factors is known to contribute to their therapeutic effect, little is known about their release of proinflammatory factors and expression of proinflammatory adhesion molecules. "Early" EPCs and "late" EPCs were isolated from human peripheral blood and their release of chemokines and thromboinflammatory mediators as well as their expression of the proinflammatory adhesion molecules was assessed at baseline and with stimulation. The effect of simvastatin on monocyte chemoattractant protein-1 (MCP-1) secretion by late EPCs from patients with vascular disease was also evaluated. All groups of EPCs released chemokines and thromboinflammatory mediators. Early EPCs primarily released thromboinflammatory mediators such as tissue factor (0.5 +/- 0.1 ng/million cells, P < 0.05), whereas adult late EPCs primarily released chemokines such as MCP-1 (287 +/- 98 ng/million cells, P < 0.05). Stimulation with tumor necrosis factor (TNF)-alpha augmented the expression of proinflammatory adhesion molecules and paracrine factors by all EPC subtypes. The release of MCP-1 by late EPCs was markedly reduced by simvastatin treatment of the cells. All EPC subtypes expressed proinflammatory paracrine factors and adhesion molecules involved in atherosclerosis. Future clinical studies should therefore not only assess the efficacy of EPCs but also monitor inflammatory activation following EPC transplantation in patients. Pharmacological modulation of EPCs before and after transplantation may represent a novel approach to improve their safety.

PINK1-associated Parkinson's disease is caused by neuronal vulnerability to calcium-induced cell death.

Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.