Stem Cell Reviews and Reports

Effects of VEGF + Mesenchymal Stem Cells and Platelet-Rich Plasma on Inbred Rat Ovarian Functions in Cyclophosphamide-Induced Premature Ovarian Insufficiency Model Abstract Premature ovarian insufficiency (POI), a fertility disorder affecting women under 40 years of age, is characterized by early loss of ovarian function. This study was aimed to maintain ovarian function in POI animal models by mesenchymal stem cells (MSCs) transplantation with/without the supplementation of platelet-rich plasma (PRP). Adipose tissue-derived MSCs were isolated from inbred rats (Fisher-344), and constitutive expression of both VEGF and GFP were maintained by transfection with plasmids, pVEGF and pGFP-N. PRP was derived from the blood of healthy untreated rats. A total of 60 rats were divided into 5 groups of 12 rats in each. First group was kept as untreated-control (Control), and POI model was induced in Fisher-344 rats by cyclophosphamide in the next four groups. Second group was kept as sham-operated-control (Sham). MSC, PRP and MSC+ PRP-treated groups were transplanted following the validation of POI model in rats. After 2 months following the transplantation, anti-mullerian-hormone (AMH) and oestradiol (E2) blood levels were measured. Follicles were evaluated after hematoxylin-eosin staining, and the immunofluorescence staining and gene expression analyses were performed to show the ovarian regeneration. The follicular count was improved after MSC- and MSC + PRP-treatment to 63% of Control-group and significantly higher than that in Sham-group, but a significant increase was not observed in PRP-group. Higher AMH and E2 levels were measured in MSC + PRP than in Sham-group, and CXCL12, BMP-4, TGF-β and IGF-1 expressions were also increased. This study showed MSCs +/-PRP transplantation after POI supports recovery of the follicular count and function. For ovarian recovery, a single administration of PRP was found not sufficient. Although MSC treatment increased follicular regeneration, better results were obtained in the co-transplantation of MSCs and PRP. These results might be promising for follicular regeneration in POI patients.

ATP-Nlrp3 Inflammasome-Complement Cascade Axis in Sterile Brain Inflammation in Psychiatric Patients and its Impact on Stem Cell Trafficking Abstract Recent evidence indicates that the occurrence of psychiatric disorders in patients is linked to a local “sterile” inflammation of brain or due to a systemic inflammation process that affects the central nervous system. This is supported by the observation that in peripheral blood of psychotic patients are detectable several mediators and markers of inflammation as well as clinical data on correlations between systemic chronic inflammatory processes and psychiatric disorders. This may explain why some reported anti-inflammatory treatment strategies have beneficial effects on ameliorating psychotic events. In this review we will present a concept that aberrant purinergic signaling and increases in extracellular level of adenosine triphosphate (ATP) in the brain parenchyma may lead to activation of Nlrp3 inflammasome in microglia cells and as a consequence microglia released danger associated molecular pattern (DAMP) proteins activate complement cascade (ComC) in mannan binding lectin (MBL) – dependent manner. Activation of ATP-Nlrp3 inflammasome-ComC axis may also orchestrate trafficking of stem cells released from bone marrow into peripheral blood observed in psychotic patients. Based on this, the ATP-Nlrp3 inflammasome-ComC axis may become a target for new therapeutic approaches, which justifies the development and clinical application of efficient anti-inflammatory treatment strategies targeting this axis in psychiatry.

Umbilical Cord Cell Therapy Improves Spatial Memory in Aging Rats Abstract There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 105 HUCPVC in 8 μl per side. The other third received 4.8 X 105 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.

Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue Abstract The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 μM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 μM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Targeting of Lung Cancer Stem Cell Self-Renewal Pathway by a Small Molecule Verrucarin J Abstract Despite considerable advances made in understanding of lung cancer biology, there has been meek improvement in lung cancer treatment outcome with 4% to 5% increase in 5-year survival rates in the last four decades. Underlying problem of lung cancer recurrence and poor prognosis is attributed to the presence of cancer stem cells (CSCs) which possess the potential to differentiate, proliferate and trigger chemo-resistance, tumor progression and metastasis, despite initial elimination of the tumor. To address specific targeting of CSCs, we investigated the effects of a small molecule Verrucarin J (VJ) on lung cancer cell lines A549 and H1793. VJ significantly inhibited cell proliferation of both cell lines, with IC50 values of approximately 10 nM for A549 and 20 nM for H1793 respectively after 48 h of treatment. A549 cell line when treated with VJ, induced cell apoptosis with concomitant down regulation of key CSC specific genes- ALDH1, LGR5, OCT4 and CD133 in a dose-dependent manner. To delineate the molecular mechanism by which VJ targets lung cancer cells and CSCs, we determined the effects of VJ on CSC self-renewal pathways Wnt1/β-catenin and Notch1. Treatment of A549 cell line with VJ inhibited significantly both the signalling pathways, suggesting inhibition of expression of CSC genes by VJ through the inhibition of CSC self-renewal signalling pathways. Taken together, our results suggest that VJ may serve as a potent anticancer drug to target cancer cells and CSCs.

Is Stem Cell Commerce in Small Animal Therapies Scientifically and Morally Justified? Abstract The lack of clear regulations for the use of veterinary stem cells has triggered the commercialization of unproven experimental therapies for companion animal diseases. Adult stem cells have complex biological characteristics that are directly related to the therapeutic application, but several questions remain to be answered. In order to regulate the use of these cells, well-conducted, controlled scientific studies that generate high-quality data should be performed, in order to assess the efficacy and safety of the intended treatment. This paper discusses the scientific challenges of mesenchymal stem cell therapy in veterinary regenerative medicine, and reviews published trials of adipose-tissue-derived stem cells in companion animal diseases that spontaneously occur.

Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties Abstract Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

Female Age Affects the Mesenchymal Stem Cell Characteristics of Aspirated Follicular Cells in the In Vitro Fertilization Programme Abstract Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31–35 years, Group C 36–39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT2 Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF Abstract While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.

Deciphering the Epitranscriptomic Signatures in Cell Fate Determination and Development Abstract Precise regulation of transcriptome modulates several vital aspects in an organism that includes gene expression, cellular activities and development, and its perturbation ensuing pathological conditions. Around 150 post-transcriptional modifications of RNA have been identified till date, which are evolutionarily conserved and likewise prevalent across RNA classes including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and detected less frequently in small nuclear RNA (snRNA) and microRNAs (miRNA). Among the RNA modifications documented, N6-methyladenosine (m6A) is the best characterised till date. Also, N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine (Ψ) are some of the other prominent modifications detected in coding and non-coding RNAs. “Epitranscriptome”, ensemble of these post-transcriptional RNA modifications, precisely coordinates gene expression and biological processes. Current literatures suggest the critical involvement of epitranscriptomics in several organisms during early development, contributing to cell fate specification and physiology. Indeed, epitranscriptomics similar to DNA epigenetics involves combinatorial dynamics provided by modified RNA molecules and associated protein complexes, which function as “writers”, “erasers” and “readers” of these modifications. A novel code orchestrating gene expression during cell fate determination is generated by the coordinated effects of different classes of modified RNAs and its regulator proteins. In this review, we summarize the current knowhow on N6-methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (ψ) modifications in RNA, the associated regulator proteins and enumerate how the epitranscriptomic regulations are involved in cell fate determination.