Human immature dental pulp stem cells` contribution to developing mouse embryos: production of human/mouse preterm chimaeras

In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Stem cells in the aetiopathogenesis and therapy of rheumatic disease

Animal models of autoimmune disease and case reports of patients with these diseases who have been involved in bone marrow transplants have provided important data implicating the haemopoietic stem cell in rheumatic disease pathogenesis. Animal and human examples exist for both cure and transfer of rheumatoid arthritis, systemic lupus erythematosus (SLE) and other organ-specific diseases using allogeneic haemopoietic stem cell transplantation. This would suggest that the stem cell in these diseases is abnormal and could be cured by replacement of a normal stem cell although more in vitro data are required in this area. Given the morbidity and increased mortality in some patients with severe autoimmune diseases and the increasing safety of autologous haemopoietic stem cell transplantation (HSCT), pilot studies have been conducted using HSCT in rheumatic diseases. It is still unclear whether an autologous graft will cure these diseases but significant remissions have been obtained which have provided important data for the design of randomized trials of HSCT versus more conventional therapy. Several trials are now open to accrual under the auspices of the European Bone Marrow Transplant Group/European League Against Rheumatism (EBMT/EULAR) registry. Future clinical and laboratory research will need to document the abnormalities of the stem cell of a rheumatic patient because new therapies based on gene therapy or stem cell differentiation could be apllied to these diseases. With increasing safety of allogeneic HSCT it is not unreasonable to predict cure of some rheumatic diseases in the near future.

Granulocyte-colony-stimulating factor (G-CSF)-primed allogeneic bone marrow: significantly less graft-versus-host disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells

Prospective studies have shown rapid engraftment using granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) for allogeneic transplantation, though the risks for graft-versus-host disease (GVHD) may be increased. It was hypothesized that the use of G-CSF to prime bone marrow (GBM) would allow rapid engraftment without increased risk for GVHD compared with G-PBSC. Patients were randomized to receive G-BM or G-PBSCs for allogeneic stem cell transplantation. The study was designed (beta < .8) to detect a difference in the incidence of chronic GVHD of 33% ( < .05). The plan was to recruit 100 patients and to conduct an interim analysis when the 6-month follow-up point was reached for the first 50 patients. Fifty-seven consecutive patients were recruited (G-BM, n = 28; G-PBSC, n = 29). Patients in the G-PBSC group received 3-fold more CD34(+) and 9-fold more CD3(+) cells. Median times to neutrophil (G-BM, 16 days; G-PBSC, 14 days; P < .1) and platelet engraftment (G-BM, 14 days; G-PBSC, 12 days; P < .1) were similar. The use of G-PBSC was associated with steroid refractory acute GVHD (G-BM, 0%; G-PBSC, 32%; P < .001), chronic GVHD (G-BM, 22%; G-PBSC, 80%; P < .02), and prolonged requirement for immunosuppressive therapy (G-BM, 173 days; G-PBSC, 680 days; P < .009). Survival was similar for the 2 groups. Compared with G-PBSC the use of G-BM resulted in comparable engraftment, reduced severity of acute GVHD, and less subsequent chronic GVHD. (Blood. 2001;98:3186-3191) (C) 2001 by The American Society of Hematology.

neptune, a Kruppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Kruppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta -globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

Objective. The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. Materials and Methods. A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. Results. We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1 HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PYlo and PYhi subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the (PYRhHolo/sp)-Rh-lo-Ho-lo PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PYhi) We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. Conclusion. Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the (RhHolosp)-Ho-lo PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Bioprocess engineering of induced pluripotent stem cells for application in cell therapy and pre-clinical research

Dissertation to obtain Master Degree in Biotechnology

Process Engineering of Stem Cells for Clinical Application

Over the last decade, human embryonic stem cells (hESCs) have garnered a lot of attention owing to their inherent self-renewal ability and pluripotency. These characteristics have opened opportunities for potential stem cell-based regenerative medicines, for development of drug discovery platforms and as unique in vitro models for the study of early human development.(...)

Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.