Derivation of human embryonic stem cells at the center for regenerative medicine in Barcelona

We report here the legislative issues related toembryo research and human embryonic stem cell (hESC)research in Spain and the derivation of nine hESC lines atthe Center of Regenerative Medicine in Barcelona. You canfind the information for obtaining our lines for researchpurposes at blc@cmrb.eu.

Coupling and monotonicity of queueing processes

The main purpose of this work is to give a survey of main monotonicity properties of queueing processes based on the coupling method. The literature on this topic is quite extensive, and we do not consider all aspects of this topic. Our more concrete goal is to select the most interesting basic monotonicity results and give simple and elegant proofs. Also we give a few new (or revised) proofs of a few important monotonicity properties for the queue-size and workload processes both in single-server and multi- server systems. The paper is organized as follows. In Section 1, the basic notions and results on coupling method are given. Section 2 contains known coupling results for renewal processes with focus on construction of synchronized renewal instants for a superposition of independent renewal processes. In Section 3, we present basic monotonicity results for the queue-size and workload processes. We consider both discrete-and continuous-time queueing systems with single and multi servers. Less known results on monotonicity of queueing processes with dependent service times and interarrival times are also presented. Section 4 is devoted to monotonicity of general Jackson-type queueing networks with Markovian routing. This section is based on the notable paper [17]. Finally, Section 5 contains elements of stability analysis of regenerative queues and networks, where coupling and monotonicity results play a crucial role to establish minimal suficient stability conditions. Besides, we present some new monotonicity results for tandem networks.

Estudi del banc de llavors d'un sòl

Treball de recerca realitzat per una alumna d'ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit cientí­fic del Jovent l'any 2009. Es tracta d'un estudi basat en les capacitats naturals regeneratives del sòl. Aquesta investigació se centra en quin grau és possible que la coberta vegetal d'unes terres que han patit pertorbacions externes (incendis, talades, etc.) pugui regenerar-se i desenvolupar-se de manera natural. De manera que, la recerca està basada en bon grau en el banc de llavors que posseeix un determinat terreny, ja que és a partir d'aquest d'on es tornarà a repoblar l'indret en qüestió. Concretament, la hipòtesi directora del treball afirma que sense la intervenció humana és possible l'aparició de nova vegetació després de l'efecte d'una pertorbació que hagi danyat totalment la coberta vegetal d'un indret. La part més experimental consisteix molt esquemàticament en realitzar diversos mostrejos a tres terrenys de diferent tipus, concretament un bosc, un conreu i un camp abandonat, per sotmetre'ls a estudi durant aproximadament cinc mesos. A partir d'aquest estudi apareixeran un seguit de conclusions que no tan sols tenen a veure amb la hipòtesi plantejada prèviament. La recerca ha conduït a conclusions incialment no tingudes en compte, però la idea principal del treball és que sí que és possible la regeneració natural de la coberta vegetal d'un indret a partir dels propàguls que aquest conté.

A general multiserver state-dependent queueing system

The work studies a general multiserver queue in which the service time of an arriving customer and the next interarrival period may depend on both the current waiting time and the server assigned to the arriving customer. Stability of the system is proved under general assumptions on the predetermined distributions describing the model. The proof exploits a combination of the Markov property of the workload process with a regenerative property of the process. The key idea leading to stability is a characterization of the limit behavior of the forward renewal process generated by regenerations. Extensions of the basic model are also studied.

Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Induced pluripotent stem (iPS) cells have generated keen interestdue to their potential use in regenerative medicine. They havebeen obtained from various cell types of both mice and humans byexogenous delivery of different combinations of Oct4, Sox2, Klf4,c-Myc, Nanog, and Lin28. The delivery of these transcription factorshas mostly entailed the use of integrating viral vectors (retrovirusesor lentiviruses), carrying the risk of both insertional mutagenesisand oncogenesis due to misexpression of these exogenousfactors. Therefore, obtaining iPS cells that do not carry integratedtransgene sequences is an important prerequisite for their eventualtherapeutic use. Here we report the generation of iPS cell linesfrom mouse embryonic fibroblasts with no evidence of integrationof the reprogramming vector in their genome, achieved by nucleofectionof a polycistronic construct coexpressing Oct4, Sox2, Klf4,and c-Myc

Generation of induced pluripotent stem cells from human cord blood cells with only two factors:OCT4 and SOX2

Induced pluripotent stem cells (iPSC ) provide an invaluable resource for regenerative medicine as they allow the generationof patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach isdesirable, such as for cell therapy of acute conditions or when the patient’s somatic cells are altered as a consequence of a chronicdisease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carryminimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousandimmunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for thederivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors(OCT 4 and SO X2) in 2 weeks and without the need for additional chemical compounds.

Generation of cardiomyocytes from new human embrionic stem cell lines derived from poor-quanlity blastocysts

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine.

Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

Smed454 dataset: unravelling the transcriptome of Schmidtea mediterranea

Background: Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. Results: In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. Conclusions: We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.

Smed454 dataset: unravelling the transcriptome of Schmidtea mediterranea

Background Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. Results In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. Conclusions We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.

La medicina regenerativa

Enguany, el premi Nobel de medicina i fisiologia és especialment significatiu per dos motius.

Insulin-Like growth-factor 1 myocardial expression decreases in chronic alcohol consumption

Background Chronic alcohol ingestion may cause severe biochemical and pathophysiological derangements to skeletal muscle. Unfortunately, these alcohol-induced events may also prime skeletal muscle for worsened, delayed, or possibly incomplete repair following acute injury. As alcoholics may be at increased risk for skeletal muscle injury, our goals were to identify the effects of chronic alcohol ingestion on components of skeletal muscle regeneration. To accomplish this, age- and gender-matched C57Bl/6 mice were provided normal drinking water or water that contained 20% alcohol (v/v) for 1820 wk. Subgroups of mice were injected with a 1.2% barium chloride (BaCl2) solution into the tibialis anterior (TA) muscle to initiate degeneration and regeneration processes. Body weights and voluntary wheel running distances were recorded during the course of recovery. Muscles were harvested at 2, 7 or 14 days post-injection and assessed for markers of inflammation and oxidant stress, fiber cross-sectional areas, levels of growth and fibrotic factors, and fibrosis. Results Body weights of injured, alcohol-fed mice were reduced during the first week of recovery. These mice also ran significantly shorter distances over the two weeks following injury compared to uninjured, alcoholics. Injured TA muscles from alcohol-fed mice had increased TNFα and IL6 gene levels compared to controls 2 days after injury. Total protein oxidant stress and alterations to glutathione homeostasis were also evident at 7 and 14 days after injury. Ciliary neurotrophic factor (CNTF) induction was delayed in injured muscles from alcohol-fed mice which may explain, in part, why fiber cross-sectional area failed to normalize 14 days following injury. Gene levels of TGFβ1 were induced early following injury before normalizing in muscle from alcohol-fed mice compared to controls. However, TGFβ1 protein content was consistently elevated in injured muscle regardless of diet. Fibrosis was increased in injured, muscle from alcohol-fed mice at 7 and 14 days of recovery compared to injured controls. Conclusions Chronic alcohol ingestion appears to delay the normal regenerative response following significant skeletal muscle injury. This is evidenced by reduced cross-sectional areas of regenerated fibers, increased fibrosis, and altered temporal expression of well-described growth and fibrotic factors.

Linear transceiver design in nonregenerative relays with channel state information

This paper deals with the design of nonregenerativerelaying transceivers in cooperative systems where channel stateinformation (CSI) is available at the relay station. The conventionalnonregenerative approach is the amplify and forward(A&F) approach, where the signal received at the relay is simplyamplified and retransmitted. In this paper, we propose an alternativelinear transceiver design for nonregenerative relaying(including pure relaying and the cooperative transmission cases),making proper use of CSI at the relay station. Specifically, wedesign the optimum linear filtering performed on the data to beforwarded at the relay. As optimization criteria, we have consideredthe maximization of mutual information (that provides aninformation rate for which reliable communication is possible) fora given available transmission power at the relay station. Threedifferent levels of CSI can be considered at the relay station: onlyfirst hop channel information (between the source and relay);first hop channel and second hop channel (between relay anddestination) information, or a third situation where the relaymay have complete cooperative channel information includingall the links: first and second hop channels and also the directchannel between source and destination. Despite the latter beinga more unrealistic situation, since it requires the destination toinform the relay station about the direct channel, it is useful as anupper benchmark. In this paper, we consider the last two casesrelating to CSI.We compare the performance so obtained with theperformance for the conventional A&F approach, and also withthe performance of regenerative relays and direct noncooperativetransmission for two particular cases: narrowband multiple-inputmultiple-output transceivers and wideband single input singleoutput orthogonal frequency division multiplex transmissions.

On regeneration and collateral sprouting to hind limb digits after sciatic nerve injury in the rat

This study examines the proportions of regenerative and collateral sprouting to the skin after peripheral nerve injury. Methods: In the first experimental paradigm, primary afferent neurones were pre-labelled with Diamidino Yellow (DY), injected in digit 3, followed by sciatic nerve section and repair. After three months of regeneration, digit 3 was re-injected with Fast Blue (FB) to label regernating cells. Fluoro-Gold (FG) was applied to the femoral (FEM) and musculocutaneous (MC) nervers four days later to quantify their contribution to the innveration. In the second experimental paradigm, sciatic nerve was first sectioned and repaired. Three months later, the sciatic was resected, and digit 3 injected with FB. After four more days, FEM and MC were resected and FG injected in all digits. Results: Neurones in dorsal root ganglion (DRG) L5 had a higher rate of correct reinnervation of digit 3 (44-72%) than neurones in DRG L4 (14-44%). Like in control cases, only occasional axons were traced from the FEM and MC. In the second experiment, only occasional labelled neurones appeared. Conclusions: The results indicate differences in the capacity for correct peripheral sensory reinnvervation between segmental levels and that in this model collateral sprouting was practically non-existent compared to regenerative sprouting.