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Biomedical subjects

Martin F Pera

Publications and source records attributed to Martin F Pera.

18 recordsLinked to original sources
PubMed2006

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system.

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.

Animals↗
PubMed2006

CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells.

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.

Biomarkers↗
PubMed2005

Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

Cell Culture Techniques↗
PubMed2005

The hESC line Envy expresses high levels of GFP in all differentiated progeny.

Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.

Base Sequence↗
PubMed2005

Derivation of neural precursors from human embryonic stem cells in the presence of noggin.

The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differentiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differentiate into astrocytes, oligodendrocytes, and mature electrophysiologically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy.

Animals↗
PubMed2005

A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells.

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM-5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM-5 is found on a 50-kDa protein present on the surface of these cells. In tissue sections of first-trimester human embryos, GCTM-5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM-5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM-5-positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM-5-positive cells that did not express cytokeratin 19 or N-CAM, classical makers of ductular reactive cells. Live GCTM-5-positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM-5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.

Adult↗
PubMed2004

Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin.

Human embryonic stem cells differentiate spontaneously in vitro into a range of cell types, and they frequently give rise to cells with the properties of extra-embryonic endoderm. We show here that endogenous signaling by bone morphogenetic protein-2 controls the differentiation of embryonic stem cells into this lineage. Treatment of embryonic stem cell cultures with the bone morphogenetic protein antagonist noggin blocks this form of differentiation and induces the appearance of a novel cell type that can give rise to neural precursors. These findings indicate that bone morphogenetic protein-2 controls a key early commitment step in human embryonic stem cell differentiation, and show that the conservation of developmental mechanisms at the cellular level can be exploited in this system--in this case, to provide a facile route for the generation of neural precursors from pluripotent cells.

Base Sequence↗
PubMed2004

Human embryonic stem cells: prospects for development.

It is widely anticipated that human embryonic stem (ES) cells will serve as an experimental model for studying early development in our species, and, conversely, that studies of development in model systems, the mouse in particular, will inform our efforts to manipulate human stem cells in vitro. A comparison of primate and mouse ES cells suggests that a common underlying blueprint for the pluripotent state has undergone significant species-specific modification. As we discuss here, technical advances in the propagation and manipulation of human ES cells have improved our understanding of their growth and differentiation, providing the potential to investigate early human development and to develop new clinical therapies.

Animals↗
PubMed2004

Presence of functional gap junctions in human embryonic stem cells.

Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription--polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.

Apoptosis↗
PubMed2003

Characterization and culture of human embryonic stem cells.

Human embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells. The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells. The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advances.

Animals↗
PubMed2002

Identification and characterisation of known and novel transcripts expressed during the final stages of human oocyte maturation.

The final stages of oocyte maturation, from the germinal vesicle (GV) stage to metaphase II (MII) oocytes, are characterised by a series of dynamic events. These include germinal vesicle break down (GVBD), resumption of meiosis, and nuclear and cytoplasmic maturation to produce MII oocytes ready for fertilisation. To investigate the specific genes transcribed during these stages of oogenesis, we have prepared and analysed amplified cDNA representing the transcribed genes in a series of GV and MII oocytes. Differential display analysis disclosed that the overall gene expression profiles between different samples of GV oocytes are very similar, regardless of their source, while those between the MII oocytes are markedly variable. A comparison of expression profiles in oocytes and somatic (cumulus) cells identified several known genes preferentially-expressed in oocytes (e.g., a zona pellucida gene), as well as five novel sequences. Two of the five novel sequences are homologous to retrotransposon sequences, long terminal repeat (LTR) and long interspersed nuclear element (LINE) 1, and two other sequences show partial homology to known ESTs and genomic sequences. The remaining sequence, which is identical to shorter ESTs isolated from germ cell tumor cDNA libraries, was extended towards its 5' end by PCR, using the original cDNA preparation from which it was isolated as a template. Expression of the resultant 1.1-kb transcript is restricted to the testis and ovary, and its expression correlates with cell pluripotency in that it is expressed in embryonal carcinoma cells, but not in their differentiated derivative cells.

Amino Acid Sequence↗
PubMed2002

Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in primate pluripotent stem cells.

We previously identified a pericellular matrix keratan sulphate/chondroitin sulphate proteoglycan present on the surface of human embryonal carcinoma stem cells, cells whose differentiation mimics early development. Antibodies reactive with various epitopes on this molecule define a cluster of differentiation markers for primate pluripotent stem cells. We describe the purification of a form of this molecule which is secreted or shed into the culture medium. Biochemical analysis of the secreted form of this molecule shows that the monomeric form, whilst containing keratan sulphate, resembles mucins in its structure and its modification with O-linked carbohydrate. Immunofluorescence and immunoblotting data show that monkey and human pluripotent stem cells react with antibodies directed against epitopes on either carbohydrate side chains or the protein core of the molecule.

Animals↗
PubMed2002

Human cloning 2001.

This review summaries human cloning from a clinical perspective. Natural human clones, that is, monozygotic twins, are increasing in the general community. Iatrogenic human clones have been produced for decades in infertile couples given fertility treatment such as ovulation induction. A clear distinction must be made between therapeutic cloning using embryonic stem cells and reproductive cloning attempts. Unlike the early clinical years of in vitro fertilization, with cloning there is no animal model that is safe and dependable. Until there is such a model, 'Dolly'-style human cloning is medically unacceptable.

Cloning, Organism↗
PubMed2002

Stem cells: hype and reality.

This update discusses what is known regarding embryonic and adult tissue-derived pluripotent stem cells, including the mechanisms underlying self-renewal without senescence, differentiation in multiple cell types both in vitro and in vivo, and future potential clinical uses of such stem cells. In Section I, Dr. Lansdorp reviews the structure and function of telomerase, the enzyme that restores telomeric ends of chromosomes upon cell division, highly present in embryonic stem cells but not adult stem cells. He discusses the structure and function of telomerase and signaling pathways activated by the enzyme, with special emphasis on normal and leukemic hematopoietic stem cells. In Section II, Dr. Pera reviews the present understanding of mammalian pluripotent embryonic stem cells. He discusses the concept of pluripotentiality in its embryonic context, derivation of stem cells from embryonic or fetal tissue, the basic properties of the stem cells, and methods to produce specific types of differentiated cell from stem cells. He examines the potential applications of stem cells in research and medicine and some of the barriers that must be crossed to achieve these goals. In Section III, Dr. Verfaillie reviews the present understanding of pluripotency of adult stem cells. She discusses the concept of stem cell plasticity, a term used to describe the greater potency described by several investigators of adult tissue-derived stem cells, critically reviews the published studies demonstrating stem cell plasticity, and possible mechanisms underlying such plasticity, and examines the possible role of pluripotent adult stem cells in research and medicine.

Animals↗