Sylvain Lehmann

Cell specific differences between human adipose-derived and mesenchymal–stromal cells despite similar differentiation potentials

Stromal cells from bone marrow and adipose tissue are attractive sources of adult progenitors for cell-based therapy. However, whether those cell populations represent intrinsically different cell types is still largely under debate. The aim of this study was to systematically and quantitatively compare adipose-derived stromal cells (ADSC) and bone marrow-derived multipotent mesenchymal-stromal cells (BM-MSC). The quantitative comparison was realized using Taqman Low Density Array, 2D electrophoresis and differentiation functional assays in vitro. Furthermore, cells engineered to express TGFbeta1 were injected into the intra-articular space of mouse knee joints in order to determine whether they were able to form new differentiated tissues in vivo. Our data revealed cell specific differences at transcriptional and proteomic levels between both cell types according to their tissue origin as well as functional differences in their differentiation processes towards adipogenic, osteogenic and chondrogenic programs. Nevertheless, in vitro as well as in vivo ADSC displayed the same ability than MSC to differentiate towards chondrocytes/osteoblasts, comforting the status of both cell sources as promising regenerative cells. In summary, our observations indicate that ADSC and MSC are fundamentally different cell types and differently committed cells.

Successful Transmission of Three Mouse-Adapted Scrapie Strains to Murine Neuroblastoma Cell Lines Overexpressing Wild-Type Mouse Prion Protein

ABSTRACT Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrPC-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrPC than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrPSc, in the agent-inoculated PrP-overexpressing cells, while no PrPSc was detectable in the untransfected cells after 3 passages. Production of PrPSc in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrPSc-A strains Chandler, 139A, and 22L but not to PrPSc-B strains 87V and 22A. We further demonstrate the successful transmission of PrPSc from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrPC and the sequence of PrPSc. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Blockade of Glycosylation Promotes Acquistion of Scrapie-like Properties by the Prion Protein in Cultured Cells

The conformational conversion of the prion protein, a sialoglycoprotein containing two N-linked oligosaccharide chains, from its normal form (PrPC) to a pathogenic form (PrPSc) is the central causative event in prion diseases. Although PrPSc can be generated in the absence of glycosylation, there is evidence that oligosaccharide chains may modulate the efficiency of the conversion process, and may also serve as molecular markers of diverse prion strains. In addition, mutational inactivation of one of the N-glycosylation sites has recently been associated with a familial spongiform encephalopathy. To investigate the role of N-glycans in determining the properties of PrP, we have expressed in transfected Chinese hamster ovary cells mouse PrP molecules in which N-glycosylation has been blocked either by treatment with the drug tunicamycin, or by substitution of alanine for threonine at one or both of the N-X-T consensus sites. We report that PrP molecules mutated at Thr182 alone or at both Thr182 and Thr196 fail to reach the cell surface after synthesis, but that those mutated at Thr196 or synthesized in the presence of tunicamycin can be detected on the plasma membrane. We also find that all three mutant PrPs, and to a limited extent wild-type PrP synthesized in the presence of inhibitor, acquire biochemical attributes reminiscent of PrPSc. We suggest that the PrP molecule has an intrinsic tendency to acquire some PrPSc-like properties, and that N-glycan chains protect against this change. However, pathogenic mutations, or presumably contact with exogenous prions, are necessary to fully convert the protein to a PrPSc state.

Oxidative stress and the prion protein in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. These diseases are believed to be the consequence of the conformational conversion of the prion protein into an abnormal isoform. Their exact pathogenic mechanism remains uncertain, but it is believed that oxidative stress plays a central role. In this article, we will first review in detail the data supporting the latter hypothesis. Subsequently, we will discuss the relationship between the prion protein and the cellular response to oxidative stress, attempting ultimately to link PrP function and neurodegeneration in these disorders.

Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival.

Many uncertainties remain regarding the physiological function of the prion protein PrP and the consequences of its conversion into the pathological scrapie isoform in prion diseases. Here, we show for the first time that different signal transduction pathways are involved in neurite outgrowth and neuronal survival elicited by PrP in cell culture of primary neurons. These pathways include the nonreceptor Src-related family member p59(Fyn), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase. Regulation of Bcl-2 and Bax expression also correlates with the survival effect elicited by PrP. The combined results, along with our observation that PrP carries the recognition molecule-related HNK-1 carbohydrate, argue strongly for a role of the molecule in neural recognition by interacting with yet unknown heterophilic neuronal receptors, as shown by comparison of neurite outgrowth from neurons of PrP-deficient and wild-type mice.

A Mutant Prion Protein Displays an Aberrant Membrane Association When Expressed in Cultured Cells

Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5. Whether membrane attachment of the mutant PrP involves integration of the polypeptide into the lipid bilayer, self-association, or binding to other membrane proteins remains to be determined. Our results suggest that alterations in the membrane association of PrP may be an important feature of prion diseases.

Recommendations to standardize preanalytical confounding factors in Alzheimer's and Parkinson's disease cerebrospinal fluid biomarkers: an update

Early diagnosis of neurodegenerative disorders such as Alzheimers (AD) or Parkinsons disease (PD) is needed to slow down or halt the disease at the earliest stage. Cerebrospinal fluid (CSF) biomarkers can be a good tool for early diagnosis. However, their use in clinical practice is challenging due to the high variability found between centers in the concentrations of both AD CSF biomarkers (Aβ42, total tau and phosphorylated tau) and PD CSF biomarker (α-synuclein). Such a variability has been partially attributed to different preanalytical procedures between laboratories, thus highlighting the need to establish standardized operating procedures. Here, we merge two previous consensus guidelines for preanalytical confounding factors in order to achieve one exhaustive guideline updated with new evidence for Aβ42, total tau and phosphorylated tau, and α-synuclein. The proposed standardized operating procedures are applicable not only to novel CSF biomarkers in AD and PD, but also to biomarkers for other neurodegenerative disorders.

Mutant and Infectious Prion Proteins Display Common Biochemical Properties in Cultured Cells

Prion diseases are unusual neurodegenerative disorders that can be both infectious and inherited. Both forms are hypothesized to result from a posttranslational structural alteration in the cell surface glycoprotein PrP (cellular isoform of the prion protein) that converts it into the protease-resistant isoform PrP (scrapie isoform of the prion protein). However, a direct comparison of molecular events underlying these two manifestations of prion diseases has not been possible, because there has been no cell culture model for the familial forms. We report here that when mutant prion proteins associated with three different inherited prion disorders of humans are expressed as their murine homologues in cultured Chinese hamster ovary cells, the proteins are protease-resistant and detergent-insoluble, two biochemical properties characteristic of infectious PrP. In addition, each mutant protein remains tightly associated with the plasma membrane after enzymatic cleavage of its glycosylphosphatidylinositol anchor, a property that we now show is also typical of infectious PrP. The cell culture system described here is the first in vitro model for familial prion diseases and provides compelling evidence that infectious and genetic cases share common molecular features.