Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells.

BACKGROUND: The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic ß-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. RESULTS: rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. CONCLUSION: Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

Emerging Roles and Potential Applications of Non-Coding RNAs in Glioblastoma.

Non-coding RNAs (ncRNAs) comprise a diversity of RNA species, which do not have the potential to encode proteins. Non-coding RNAs include two classes of RNAs, namely: short regulatory ncRNAs and long non-coding RNAs (lncRNAs). The short regulatory RNAs, containing up to 200 nucleotides, include small RNAs, such as microRNAs (miRNA), short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). The lncRNAs include long antisense RNAs and long intergenic RNAs (lincRNAs). Non-coding RNAs have been implicated as master regulators of several biological processes, their expression being strictly regulated under physiological conditions. In recent years, particularly in the last decade, substantial effort has been made to investigate the function of ncRNAs in several human diseases, including cancer. Glioblastoma is the most common and aggressive type of brain cancer in adults, with deregulated expression of small and long ncRNAs having been implicated in onset, progression, invasiveness, and recurrence of this tumor. The aim of this review is to guide the reader through important aspects of miRNA and lncRNA biology, focusing on the molecular mechanism associated with the progression of this highly malignant cancer type.

Extracellular matrix dynamics during mesenchymal stem cells differentiation.

Mesenchymal stem cells (MSCs) are stromal cells that display self-renewal and multipotent differentiation capacity. The repertoire of mature cells generated ranges but is not restricted to: fat, bone and cartilage. Their potential importance for both cell therapy and maintenance of in vivo homeostasis is indisputable. Nonetheless, both their in vivo identity and use in cell therapy remain elusive. A drawback generated by this fact is that little is known about the MSC niche and how it impacts differentiation and homeostasis maintenance. Hence, the roles played by the extracellular matrix (ECM) and its main regulators namely: the Matrix Metalloproteinases (MMPs) and their counteracting inhibitors (TIMPs and RECK) upon stem cells differentiation are only now beginning to be unveiled. Here, we will focus on mesenchymal stem cells and review the main mechanisms involved in adipo, chondro and osteogenesis, discussing how the extracellular matrix can impact not only lineage commitment, but, also, their survival and potentiality. This review critically analyzes recent work in the field in an effort towards a better understanding of the roles of Matrix Metalloproteinases and their inhibitors in the above-cited events.

Spatio-temporal expression profile of matrix metalloproteinase (Mmp) modulators Reck and Sparc during the rat ovarian dynamics.

BACKGROUND: Matrix metalloproteinases (Mmps) and their tissue inhibitors (Timps) are widely recognized as crucial factors for extracellular matrix remodeling in the ovary and are involved in follicular growth, ovulation, luteinization, and luteolysis during the estrous cycle. Recently, several genes have been associated to the modulation of Mmps activity, including Basigin (Bsg), which induces the expression of Mmps in rat ovaries; Sparc, a TGF-ß modulator that is related to increased expression of Mmps in cancer; and Reck, which is associated with Mmps inhibition. However, the expression pattern of Mmp modulators in ovary dynamics is still largely uncharacterized. METHODS: To characterize the expression pattern of Mmps network members in ovary dynamics, we analyzed the spatio-temporal expression pattern of Reck and Sparc, as well as of Mmp2, Mmp9 and Mmp14 proteins, by immunohistochemistry (IHC), in pre-pubertal rat ovaries obtained from an artificial cycle induced by eCG/hCG, in the different phases of the hormone-induced estrous cycle. We also determined the gene expression profiles of Mmps (2, 9, 13 14), Timps (1, 2, 3), Sparc, Bsg, and Reck to complement this panel. RESULTS: IHC analysis revealed that Mmp protein expression peaks at the early stages of folliculogenesis and ovulation, decreases during ovulation-luteogenesis transition and luteogenesis, increasing again during corpus luteum maintenance and luteolysis. The protein expression patterns of these metalloproteinases and Sparc were inverse relative to the pattern displayed by Reck. We observed that the gene expression peaks of Mmps inhibitors Reck and Timp2 were closely paraleled by Mmp2 and Mmp9 suppression. The opposite was also true: increased Mmp2 and Mmp9 expression was concomitant to reduced Reck and Timp2 levels. CONCLUSION: Therefore, our results generate a spatio-temporal expression profile panel of Mmps and their regulators, suggesting that Reck and Sparc seem to play a role during ovarian dynamics: Reck as a possible inhibitor and Sparc as an inducer of Mmps.

Existence of a potential neurogenic system in the adult human brain.

BACKGROUND: Prevailingly, adult mammalian neurogenesis is thought to occur in discrete, separate locations known as neurogenic niches that are best characterized in the subgranular zone (SGZ) of the dentate gyrus and in the subventricular zone (SVZ). The existence of adult human neurogenic niches is controversial. METHODS: The existence of neurogenic niches was investigated with neurogenesis marker immunostaining in histologically normal human brains obtained from autopsies. Twenty-eight adult temporal lobes, specimens from limbic structures and the hypothalamus of one newborn and one adult were examined. RESULTS: The neural stem cell marker nestin stained circumventricular organ cells and the immature neuronal marker doublecortin (DCX) stained hypothalamic and limbic structures adjacent to circumventricular organs; both markers stained a continuous structure running from the hypothalamus to the hippocampus. The cell proliferation marker Ki-67 was detected predominantly in structures that form the septo-hypothalamic continuum. Nestin-expressing cells were located in the fimbria-fornix at the insertion of the choroid plexus; ependymal cells in this structure expressed the putative neural stem cell marker CD133. From the choroidal fissure in the temporal lobe, a nestin-positive cell layer spread throughout the SVZ and subpial zone. In the subpial zone, a branch of this layer reached the hippocampal sulcus and ended in the SGZ (principally in the newborn) and in the subiculum (principally in the adults). Another branch of the nestin-positive cell layer in the subpial zone returned to the optic chiasm. DCX staining was detected in the periventricular and middle hypothalamus and more densely from the mammillary body to the subiculum through the fimbria-fornix, thus running through the principal neuronal pathway from the hippocampus to the hypothalamus. The column of the fornix forms part of this pathway and appears to coincide with the zone previously identified as the human rostral migratory stream. Partial co-labeling with DCX and the neuronal marker ßIII-tubulin was also observed. CONCLUSIONS: Collectively, these findings suggest the existence of an adult human neurogenic system that rises from the circumventricular organs and follows, at minimum, the circuitry of the hypothalamus and limbic system.

Bone Morphogenetic Proteins: structure, biological function and therapeutic applications.

Bone Morphogenetic Proteins (BMPs) are multifunctional secreted cytokines, which belong to the TGF-ß superfamily. These glycoproteins act as a disulfide-linked homo- or heterodimers, being potent regulators of bone and cartilage formation and repair, cell proliferation during embryonic development and bone homeostasis in the adult. BMPs are promising molecules for tissue engineering and bone therapy. The present review discusses this family of proteins, their structure and biological function, their therapeutic applications and drawbacks, their effects on mesenchymal stem cells differentiation, and the cell signaling pathways involved in this process.

Recombinant Human Peptide Growth Factors, Bone Morphogenetic Protein-7 (rhBMP7), and Platelet-Derived Growth Factor-BB (rhPDGF-BB) for Osteoporosis Treatment in an Oophorectomized Rat Model.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate/stimulate osteogenesis, playing vital roles in bone homeostasis, rendering them strong candidates for osteoporosis treatment. We evaluated the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in an oophorectomy-induced osteoporosis rat model. Forty Sprague Dawley rats underwent oophorectomy surgery; treatments commenced on the 100th day post-surgery when all animals exhibited signs of osteoporosis. These peptide growth factors were administered intraocularly (iv) once or twice a week and the animals were monitored for a total of five weeks. Two weeks after the conclusion of the treatments, the animals were euthanized and tissues were collected for assessment of alkaline phosphatase, X-ray, micro-CT, and histology. The results indicate that the most promising treatments were 20 µg/kg rhPDGF-BB + 30 µg/kg rhBMP-7 twice a week and 30 µg/kg BMP-7 twice a week, showing significant increases of 15% (p < 0.05) and 13% (p < 0.05) in bone volume fraction and 21% (p < 0.05) and 23% (p < 0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 have demonstrated the ability to increase bone volume and density in this osteoporotic animal model, establishing them as potential candidates for osteoporosis treatment.

Anatomic and Surgical Aspects of Total Pancreatectomy in Swine.

BACKGROUND: The swine is a valuable model for preclinical research and surgical technique training. Induction of Type I diabetes is achieved by total pancreatectomy, therefore these animals may be used in several research studies, including islet transplantation field. Given the lack of information in the literature, the purpose of this work is to describe anatomic aspects of swine pancreas, the total pancreatectomy surgical technique, intra- and postoperative complications and the autopsy results. MATERIAL AND METHODS: Five hybrid male pigs, 20-35 kg, submitted to total pancreatectomy with duodenum, bile duct, and spleen preservation. Postoperatively, daily clinical assessment and capillary blood glucose collection were performed. At the end of the 30-day period or in the occurrence of serious clinical complications, euthanasia and autopsy were performed. RESULTS: The average duration of surgery was 128 minutes, without intraoperative deaths or anesthesia induction failures. The median survival was 6.6 days. Postoperative complications were weight loss (3), emesis (2), constipation (2), abdominal distension (2), diarrhea (1), and loss of appetite (1). All animals were euthanized due to serious complications. Two animals presented surgical complications (duodenal necrosis with gastroparesis and internal hernia with intestinal necrosis). The other 3 animals presented serious clinical complications related to exocrine pancreatic insufficiency due to deficiency of pancreatic enzymes. Glycemic values above 200 mg/dL were found on the first postoperative day and above 300 mg/dL on the seventh day in all animals. CONCLUSION: A model of total pancreatectomy with duodenum, spleen, and bile duct preservation in pigs was established. All animals became diabetic, however, animals without postoperative complications were euthanized due to serious complications related to pancreas exocrine insufficiency.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells.

BACKGROUND: Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. RESULTS: From 150 µg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. CONCLUSIONS: Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

Islet transplantation: overcoming the organ shortage.

BACKGROUND: Type 1 diabetes mellitus (T1D) is a condition resulting from autoimmune destruction of pancreatic ß cells, leading patients to require lifelong insulin therapy, which, most often, does not avoid the most common complications of this disease. Transplantation of isolated pancreatic islets from heart-beating organ donors is a promising alternative treatment for T1D, however, this approach is severely limited by the shortage of pancreata maintained under adequate conditions. METHODS: In order to analyze whether and how this problem could be overcome, we undertook a retrospective study from January 2007 to January 2010, evaluating the profile of brain-dead human pancreas donors offered to our Cell and Molecular Therapy NUCEL Center ( www.usp.br/nucel ) and the basis for organ refusal. RESULTS: During this time period, 558 pancreata were offered by the São Paulo State Transplantation Central, 512 of which were refused and 46 were accepted for islet isolation and transplantation. Due to the elevated number of refused organs, we decided to analyze the main reasons for refusal in order to evaluate the possibility of improving the organ acceptance rate. The data indicate that hyperglycemia, technical issues, age, positive serology and hyperamylasemia are the top five main causes for declination of a pancreas offer. CONCLUSIONS: This study underlines the main reasons to decline a pancreas offer in Sao Paulo-Brazil and provides some guidance to ameliorate the rate of eligible pancreas donors, aiming at improving the islet isolation and transplantation outcome. TRIAL REGISTRATION: Protocol CAPPesq number 0742/02/CONEP 9230.

Role of MSC-derived small extracellular vesicles in tissue repair and regeneration.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and repair, secreting vesicles to the extracellular environment. Isolated exosomes were shown to affect angiogenesis, immunomodulation and tissue regeneration. Numerous efforts have been dedicated to describe the mechanism of action of these extracellular vesicles (EVs) and guarantee their safety, since the final aim is their therapeutic application in the clinic. The major advantage of applying MSC-derived EVs is their low or inexistent immunogenicity, prompting their use as drug delivery or therapeutic agents, as well as wound healing, different cancer types, and inflammatory processes in the neurological and cardiovascular systems. MSC-derived EVs display no vascular obstruction effects or apparent adverse effects. Their nano-size ensures their passage through the blood-brain barrier, demonstrating no cytotoxic or immunogenic effects. Several in vitro tests have been conducted with EVs obtained from different sources to understand their biology, molecular content, signaling pathways, and mechanisms of action. Application of EVs to human therapies has recently become a reality, with clinical trials being conducted to treat Alzheimer's disease, retina degeneration, and COVID-19 patients. Herein, we describe and compare the different extracellular vesicles isolation methods and therapeutic applications regarding the tissue repair and regeneration process, presenting the latest clinical trial reports.

Stem cells differentiation into insulin-producing cells (IPCs): recent advances and current challenges.

Type 1 diabetes mellitus (T1D) is a chronic disease characterized by an autoimmune destruction of insulin-producing ß-pancreatic cells. Although many advances have been achieved in T1D treatment, current therapy strategies are often unable to maintain perfect control of glycemic levels. Several studies are searching for new and improved methodologies for expansion of ß-cell cultures in vitro to increase the supply of these cells for pancreatic islets replacement therapy. A promising approach consists of differentiation of stem cells into insulin-producing cells (IPCs) in sufficient number and functional status to be transplanted. Differentiation protocols have been designed using consecutive cytokines or signaling modulator treatments, at specific dosages, to activate or inhibit the main signaling pathways that control the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic ß-cells. Here, we provide an overview of the current approaches and achievements in obtaining stem cell-derived ß-cells and the numerous challenges, which still need to be overcome to achieve this goal. Clinical translation of stem cells-derived ß-cells for efficient maintenance of long-term euglycemia remains a major issue. Therefore, research efforts have been directed to the final steps of in vitro differentiation, aiming at production of functional and mature ß-cells and integration of interdisciplinary fields to generate efficient cell therapy strategies capable of reversing the clinical outcome of T1D.

The Influence of rhBMP-7 Associated with Nanometric Hydroxyapatite Coatings Titanium Implant on the Osseointegration: A Pre-Clinical Study.

Background: Bioceramic nanometer coatings have been regarded as potential substitutes for plasma-sprayed hydroxyapatite coatings, and the association with bone morphogenetic protein (BMP) is an attempt to achieve faster osseointegration to hasten oral rehabilitation. Objective: This study aimed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the osseointegration of titanium implants coated with a thin film surface of hydroxyapatite (HA). Methods: Two implants (n = 24) were placed in each white New Zealand rabbits' femur (n = 6). Implants were placed in the right femur after standard instrumentation (A and B) and in the left femur after an over-instrumentation (C and D), preventing bone-implant contact. The distal implants were installed associated with rhBMP-7 (groups B [regular instrumentation] and D [over-instrumentation]) and, also, in the absence of without BMP (control groups A [regular instrumentation] and C [over-instrumentation]). After 4 weeks, the animals were euthanized. The bone blocks containing the implants were embedded in methyl methacrylate and sectioned parallel to the long axis of the implant, which were analyzed by image segmentation. The data were analyzed using a nonparametric statistical method. Results: We observed that Group A had a mean bone formation of 35.6% compared to Group B, which had 48.6% (p > 0.05). Moreover, this group showed 28.3% of connective tissue compared to Group A, with 39.3%. In the over-instrumented groups, rhBMP-7 (Group D) showed an enhanced and significant increase in bone formation when compared with the group without rhBMP-7 (Group C). Conclusion: We concluded that the association of rhBMP-7 to thin nanostructure HA-coated implants promoted greater new bone area than the same implants in the absence of rhBMP-7, mainly in cases of over-instrumented implant sites.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS.

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

R-Spondin1 enhances wnt signaling and decreases weight loss in short bowel syndrome zebrafish.

BACKGROUND: R-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/ß-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA. METHODS: Adult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis. RESULTS: Repeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for ß-catenin and gene expression for cyclin D1 was increased. CONCLUSIONS: Intraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased ß-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway.

Kidney organoids generated from erythroid progenitors cells of patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis. METHODS: Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. RESULTS: iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. CONCLUSION: The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Characterization of rat liver bud-derived cells.

Cells derived from the fetal liver have been shown to be a rich source of progenitor stem cells, constituting a promising source for Tissue Engineering and Regenerative Medicine. In this study, embryo and fetal liver-bud derived cells from Fischer 344 rats were obtained at E12.5, E14.5 and E16.5 gestational days and evaluated for cell phenotype, survival and proliferation. Liver transaminase (AST and ALT) and AFP levels were lower in embryo liver-bud-derived cells on day 12.5. Markers for stem cells, cell cycle progression and cell death were differentially expressed in E12.5 cell cultures. Analysis of mitochondrial electric potential on 14.5 and 16.5 days showed a tendency for cells with lower functional or metabolic ability, in comparison to cultures derived from day 12.5. The results demonstrated that the majority of the E16.5 cells were in the G0 / G1 phase. The capacity of synthesis (S) and cellular division (G2 / M) of embryo and fetal liver bud-derived cells was constant over all gestational periods. In conclusion, embryo and fetal liver-bud-derived cells during the periods of 12.5 and 14.5 days, showed expression profile of progenitor cells, cell activity and hematopoietic function in culture.

Low RECK Expression Is Part of the Cervical Carcinogenesis Mechanisms.

Human papillomavirus (HPV)-induced carcinogenesis comprises alterations in the expression and activity of matrix metalloproteinases (MMP) and their regulators. Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) inhibits the activation of specific metalloproteinases and its expression is frequently lost in human cancers. Here we analyzed the role of RECK in cervical carcinogenesis. Cervical cancer derived cell lines over expressing RECK were used to determine tumor kinetics as well as, cellular, immune and molecular properties in vivo. Besides, we analyzed RECK expression in cervical cancer samples. RECK over expression (RECK+) delayed tumor growth and increased overall survival in vivo. RECK+ tumors displayed an increase in lymphoid-like inflammatory infiltrating cells, reduced number and viability of tumor and endothelial cells and lower collagenase activity. RECK+ tumors exhibited an enrichment of cell adhesion processes both in the mouse model and cervical cancer clinical samples. Finally, we found that lower RECK mRNA levels were associated with cervical lesions progression and worse response to chemotherapy in cervical cancer patients. Altogether, we show that increased RECK expression reduced the tumorigenic potential of HPV-transformed cells both in vitro and in vivo, and that RECK down regulation is a consistent and clinically relevant event in the natural history of cervical cancer.

Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9.

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.