Isolation and characterisation of mouse amniotic fluid stem cells: study of their origin, regenerative potential and reprogramming into pluripotent cells

Introduction: Stem cells are defined by their ability to proliferate for a long period of time, a property known as ‘self-renewal’, and to give rise to differentiated cells. Stem cells can be distinguished into totipotent, pluripotent, multipotent, oligopotent and unipotent. They can also be classified into embryonic, adult and fetal stem cells. Embryonic stem (ES) cells are obtained from inner cell mass (ICM) of blastocyst and are puripotent. Primordial germ cells (PGC) in the embryo give rise to gametes but they are not pluripotent, albeit they express Oct4, Nanog and Sox2. They can be reprogrammed in vitro, becoming pluripotent embryonic germ (EG) cells. Amniotic fluid stem (AFS) cells are fetal stem cells that can be isolated from the amniotic fluid (AF) by the expression of the marker c-kit, both in human and mouse, but their origin is unknown. Human AFS are multipotent in vitro, while both human and mouse AFS have hematopoitic potential, in vitro and in vivo. Recently it has been demonstrated that human AFS from first and second thrimester can be reprogrammend into pluripotent cells in vitro, after supplementation with Valproic Acid (VPA). Cells from the AF have also been used to obtain induced pluripotent stem (iPS) cells. For all these reasons AFS cells seems to be a promising sources of cells for regenerative medicine. Spinal muscular atrophy (SMA) is an autosomal recessive disease, caused by an homozygous deletion or mutation of the motor neuron 1 (SMN1) gene. Bone marrow (BM) transplantation in a murine model of SMA attenuates the myopathic phenotype without a full recovery and without long-term therapeutic effects. Aims of the thesis: Characterisation of fresh mouse AFS cells, evaluation of their myogenic potential into a model of SMA (HSA-Cre, SmnF7/F7 mouse) investigation of their putative PGC origin and induction to pluripotency through a non-viral method (PiggyBac, PB). Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected as c-kit+ cells with magnetic beads. Freshly isolated-AFS cells were analized for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, Sca1, CD105) by flow citometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. Hematopoitic potential was evaluated in vitro, while the teratoma assay was performed in Rag2-/-γc-/- mice. For the reprogramming into EG cells cells were seeded into a feeder layer of mitotically inactivated STO or Sl4-m220, in a PGC medium supplemented with LIF and basic fibroblast growth factor (bFGF) and in N2B27 2iLIF medium. For the treatment of HSA-Cre, SmnF7/F7 mice, GFP+ cells were injected via the tail vain and sacrificed one month after transplantation. Tibialis Anteriour (TA) muscles were stained with hematoxylin and eosin, Masson’s trichrome and analized by immunofluorescence for positivity for GFP/dystrophin. The experiments for the origin of AFS have been conducted using two mouse models: Oct4-GFP and TNAP-Cre. For the induction to pluripotency cells were obtanied from Oct4-GFP positive embryos, and transfected with the PB-TET transposon plasmid containing four genes (Oct-4, Sox-2, c-Myc and Klf4) under the transcriptional control of the tetO2 tetracycline/doxycycline inducible promoter. The expression of pluripotency gene was induced with doxycycline. iPS cells obtained were tested for the expression of Nanog, SSEA-1 and for positivity to alkaline phosphatase. Results: Mouse AFS number chaged during the course of gestation. These cells expressed hematopoietic markers (CD45, CD34, Sca1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. On the basis of c-kit expression two populations were defined: c-kithigh and c-kitlow which showed differential expression of the aforementioned markers. c-kitlow are the more abundant, but during the course of gestation they decreases in numbers while the number of c-kithigh cells increases. Both populations had hematopoietic potential vitro. Gene expression analysis showed that mouse AFS cells expressed at low levels Oct4 and Sox2 and high levels c-Myc and Klf4, and their expression changed during the course of gestation. Single cell PCR showed that at E13.5 there 5% of cells co-expressed Oct4, Sox2 and Klf4. Mouse AFS cells didn't form teratoma. In the cell therapy experiments HSA-Cre, SmnF7/F7 control mice died at the age of 10 months, while mice treated with GFP+ AFS or bone marrow (BM) cells had a survival rates increased by 75% and 50% respectively. HSA-Cre, SmnF7/F7 mice treated with AFS cells recovered more than 75% of force compared to the untreated animals. One month after transplantation, muscles from AFS-treated mice displayed very low number of regenerating myofibers (<1%) and normal dystrophin expression; moreover, 37.86% (± 9.48%) of the fibers were GFP+. 15 months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a mild-phenotype, close to wild-type mice, and 58.00% (± 2.43%) of the myofibers were GFP+. Similar results were obtained with HSA-Cre, SmnF7/F7 treated with mouse AFS cells expanded in culture. To evaluate if mouse AFS cells were PGC cells, they were cultivated following the protocol established to obtain pluripotent EG cells from PGC cells. Two different culture protocols were used, but no EG cells were obtained. AFS cells isolated from Oct4-GFP fetuses at different embryonic stages showed no presence of Oct4+ cells. The TNAP-Cre line resulted to be unspecific. iPS clones obtained transfecting mouse AFS cells were doxycycline indipendent, they expressed Oct4, they were positive for Nanog and SSEA1, and for the alkaline phosphatase. Discussion: Mouse AFS cells are an heterogenous population, and their phenotype changed during the course of gestation. They expressed mesenchymal, hematopietic and endothelial markers. The two populations (c-kithigh and c-kitlow) should be tested in vivo to asses their differentiative potential. Gene expression analysis at population and single cells level confirmed the heterogeinity of mouse AFS cells. AFS showed a myogenic potential, even after long-term transplantion, suggesting an interesting therapeutic potential of these cells. AFS could contribute to the formation of new myofibers by fusing with existing ones or after integration within the stem cell niche of the muscle. The study of their origin suggested that mouse AFS cells aren't PGC. However it is important to remind that the Oct4-GFP mouse is not a lineage-tracking model; therefore more experiments are needed to confirm these results and to find the origin of these cells. iPS cells are a promising research tool to obtain a model of several diseases or as a source of cells for therapeutic approaches. Here it has been shown that the PB system is a suitable method for the reprogramming of mouse AFS cells. These are only preliminary results and more experiments will be necessary to complete the characterisation of these cells

First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration

Applicability of in vivo staging of regional amyloid burden in a cognitively normal cohort with subjective memory complaints: the INSIGHT-preAD study.

BACKGROUND:Current methods of amyloid PET interpretation based on the binary classification of global amyloid signal fail to identify early phases of amyloid deposition. A recent analysis of 18F-florbetapir PET data from the Alzheimer's disease Neuroimaging Initiative cohort suggested a hierarchical four-stage model of regional amyloid deposition that resembles neuropathologic estimates and can be used to stage an individual's amyloid burden in vivo. Here, we evaluated the validity of this in vivo amyloid staging model in an independent cohort of older people with subjective memory complaints (SMC). We further examined its potential association with subtle cognitive impairments in this population at elevated risk for Alzheimer's disease (AD). METHODS:The monocentric INSIGHT-preAD cohort includes 318 cognitively intact older individuals with SMC. All individuals underwent 18F-florbetapir PET scanning and extensive neuropsychological testing. We projected the regional amyloid uptake signal into the previously proposed hierarchical staging model of in vivo amyloid progression. We determined the adherence to this model across all cases and tested the association between increasing in vivo amyloid stage and cognitive performance using ANCOVA models. RESULTS:In total, 156 participants (49%) showed evidence of regional amyloid deposition, and all but 2 of these (99%) adhered to the hierarchical regional pattern implied by the in vivo amyloid progression model. According to a conventional binary classification based on global signal (SUVRCereb = 1.10), individuals in stages III and IV were classified as amyloid-positive (except one in stage III), but 99% of individuals in stage I and even 28% of individuals in stage II were classified as amyloid-negative. Neither in vivo amyloid stage nor conventional binary amyloid status was significantly associated with cognitive performance in this preclinical cohort. CONCLUSIONS:The proposed hierarchical staging scheme of PET-evidenced amyloid deposition generalizes well to data from an independent cohort of older people at elevated risk for AD. Future studies will determine the prognostic value of the staging approach for predicting longitudinal cognitive decline in older individuals at increased risk for AD

Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia

open12siopenSchiavo, Andrea Alex; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, Gian Paolo; De Coppi, Paolo; Pozzobon, MichelaSchiavo, ANDREA ALEX; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, GIAN PAOLO; DE COPPI, Paolo; Pozzobon, Michel

Isolation and characterisation of mouse amniotic fluid stem cells: study of their origin, regenerative potential and reprogramming into pluripotent cells

Introduction: Stem cells are defined by their ability to proliferate for a long period of time, a property known as ‘self-renewal’, and to give rise to differentiated cells. Stem cells can be distinguished into totipotent, pluripotent, multipotent, oligopotent and unipotent. They can also be classified into embryonic, adult and fetal stem cells. Embryonic stem (ES) cells are obtained from inner cell mass (ICM) of blastocyst and are puripotent. Primordial germ cells (PGC) in the embryo give rise to gametes but they are not pluripotent, albeit they express Oct4, Nanog and Sox2. They can be reprogrammed in vitro, becoming pluripotent embryonic germ (EG) cells. Amniotic fluid stem (AFS) cells are fetal stem cells that can be isolated from the amniotic fluid (AF) by the expression of the marker c-kit, both in human and mouse, but their origin is unknown. Human AFS are multipotent in vitro, while both human and mouse AFS have hematopoitic potential, in vitro and in vivo. Recently it has been demonstrated that human AFS from first and second thrimester can be reprogrammend into pluripotent cells in vitro, after supplementation with Valproic Acid (VPA). Cells from the AF have also been used to obtain induced pluripotent stem (iPS) cells. For all these reasons AFS cells seems to be a promising sources of cells for regenerative medicine. Spinal muscular atrophy (SMA) is an autosomal recessive disease, caused by an homozygous deletion or mutation of the motor neuron 1 (SMN1) gene. Bone marrow (BM) transplantation in a murine model of SMA attenuates the myopathic phenotype without a full recovery and without long-term therapeutic effects. Aims of the thesis: Characterisation of fresh mouse AFS cells, evaluation of their myogenic potential into a model of SMA (HSA-Cre, SmnF7/F7 mouse) investigation of their putative PGC origin and induction to pluripotency through a non-viral method (PiggyBac, PB). Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected as c-kit+ cells with magnetic beads. Freshly isolated-AFS cells were analized for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, Sca1, CD105) by flow citometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. Hematopoitic potential was evaluated in vitro, while the teratoma assay was performed in Rag2-/-γc-/- mice. For the reprogramming into EG cells cells were seeded into a feeder layer of mitotically inactivated STO or Sl4-m220, in a PGC medium supplemented with LIF and basic fibroblast growth factor (bFGF) and in N2B27 2iLIF medium. For the treatment of HSA-Cre, SmnF7/F7 mice, GFP+ cells were injected via the tail vain and sacrificed one month after transplantation. Tibialis Anteriour (TA) muscles were stained with hematoxylin and eosin, Masson’s trichrome and analized by immunofluorescence for positivity for GFP/dystrophin. The experiments for the origin of AFS have been conducted using two mouse models: Oct4-GFP and TNAP-Cre. For the induction to pluripotency cells were obtanied from Oct4-GFP positive embryos, and transfected with the PB-TET transposon plasmid containing four genes (Oct-4, Sox-2, c-Myc and Klf4) under the transcriptional control of the tetO2 tetracycline/doxycycline inducible promoter. The expression of pluripotency gene was induced with doxycycline. iPS cells obtained were tested for the expression of Nanog, SSEA-1 and for positivity to alkaline phosphatase. Results: Mouse AFS number chaged during the course of gestation. These cells expressed hematopoietic markers (CD45, CD34, Sca1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. On the basis of c-kit expression two populations were defined: c-kithigh and c-kitlow which showed differential expression of the aforementioned markers. c-kitlow are the more abundant, but during the course of gestation they decreases in numbers while the number of c-kithigh cells increases. Both populations had hematopoietic potential vitro. Gene expression analysis showed that mouse AFS cells expressed at low levels Oct4 and Sox2 and high levels c-Myc and Klf4, and their expression changed during the course of gestation. Single cell PCR showed that at E13.5 there 5% of cells co-expressed Oct4, Sox2 and Klf4. Mouse AFS cells didn't form teratoma. In the cell therapy experiments HSA-Cre, SmnF7/F7 control mice died at the age of 10 months, while mice treated with GFP+ AFS or bone marrow (BM) cells had a survival rates increased by 75% and 50% respectively. HSA-Cre, SmnF7/F7 mice treated with AFS cells recovered more than 75% of force compared to the untreated animals. One month after transplantation, muscles from AFS-treated mice displayed very low number of regenerating myofibers (<1%) and normal dystrophin expression; moreover, 37.86% (± 9.48%) of the fibers were GFP+. 15 months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a mild-phenotype, close to wild-type mice, and 58.00% (± 2.43%) of the myofibers were GFP+. Similar results were obtained with HSA-Cre, SmnF7/F7 treated with mouse AFS cells expanded in culture. To evaluate if mouse AFS cells were PGC cells, they were cultivated following the protocol established to obtain pluripotent EG cells from PGC cells. Two different culture protocols were used, but no EG cells were obtained. AFS cells isolated from Oct4-GFP fetuses at different embryonic stages showed no presence of Oct4+ cells. The TNAP-Cre line resulted to be unspecific. iPS clones obtained transfecting mouse AFS cells were doxycycline indipendent, they expressed Oct4, they were positive for Nanog and SSEA1, and for the alkaline phosphatase. Discussion: Mouse AFS cells are an heterogenous population, and their phenotype changed during the course of gestation. They expressed mesenchymal, hematopietic and endothelial markers. The two populations (c-kithigh and c-kitlow) should be tested in vivo to asses their differentiative potential. Gene expression analysis at population and single cells level confirmed the heterogeinity of mouse AFS cells. AFS showed a myogenic potential, even after long-term transplantion, suggesting an interesting therapeutic potential of these cells. AFS could contribute to the formation of new myofibers by fusing with existing ones or after integration within the stem cell niche of the muscle. The study of their origin suggested that mouse AFS cells aren't PGC. However it is important to remind that the Oct4-GFP mouse is not a lineage-tracking model; therefore more experiments are needed to confirm these results and to find the origin of these cells. iPS cells are a promising research tool to obtain a model of several diseases or as a source of cells for therapeutic approaches. Here it has been shown that the PB system is a suitable method for the reprogramming of mouse AFS cells. These are only preliminary results and more experiments will be necessary to complete the characterisation of these cells.Introduzione: Le cellule staminali hanno la capacità di dare orgine ad una progenie di cellule mature mantendo la capacità di autorinnovamento. Possono essere distinte sulla base delle loro potenzialità in totipotenti, pluripotenti, multipotenti, oligopotenti e unipotenti. Possono anche essere distinte in cellule staminali embrionali, fetali e adulte. Le cellule staminali embrionali, ottenute dalla massa cellulare interna della blastocisti, sono pluripotenti. Nell'embrione le cellule primordiali germinali danno origine ai gameti, ed esprimono i marcatori di pluripotenza (Oct4, Nanog, Sox2), ma non sono pluripotenti. Esse possono essere riprogrammate in vitro, diventando così cellule germinali embrionali. Tra le cellule staminali fetali ci sono le cellule staminali del liquido amniotico (AFS). Queste cellule sono isolate dal liquido amniotico per la positività al marcatore c-kit e sono presenti sia nell'uomo che nel topo, anche se la loro origine embrionale non è nota. Le cellule AFS umane sono multipotenti in vitro; le cellule AFS umane e murine hanno uno specifico potenziale ematopoietico, in vivo e in vitro. Recentemente è stato dimostrato che le cellule AFS umane ottenute dal primo e dal secondo trimestre di gravidanza, possono essere riprogrammate in vitro in cellule pluripotenti, a seguito dell'aggiunta di acido valproico. Inoltre, le cellule del liquido amniotico sono state anche utilizzate da diversi gruppi di ricerca per ottenere cellule staminali indotte alla pluripotenza (iPS). Per questi motivi, le AFS rappresentano una sorgente innovativa di cellule per approcci di medicina rigenerativa. L'atrofia spinale muscolare (SMA) è una malattia autosomica recessiva, causata della delezione o mutazione omozigote del gene della sopravvivvenza del motoneurone 1 (SMN1). Il trapianto di midollo osseo in un modello murino di SMA attenua il fenotipo miopatico, tuttavia non lo recupera totalmente e non mostra alcun effetto benefico a lungo termine. Scopo della tesi: Gli scopi di questa tesi consistono nella caratterizazzione delle cellule murine AFS isolate a fresco, nella valutaione del loro potenziale miogenico dopo il trapianto in animali HSA-Cre, SmnF7/F7, nello studio della loro origine embrionale e nell'induzione alla pluripotenza usando un metodo non virale (PiggyBac, PB). Materiali e Metodi: Le cellule murine AFS sono state ottenute attraverso amniocentesi e successiva immunoselezione per il marcatore c-kit mediante biglie magnetiche. Le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, Sca1, CD105) attraverso la citometria a flusso e per l'espressione di Oct4, Sox2, c-Myc, Klf4, Nanog e Sca-1, attraverso qRT-PCR , a diversi stadi embrionali. Sono stati valutati il potenziale ematopoietico in vitro e la capacità di formare teratomi in topi Rag2-/-γc-/-. Per la riprogrammazione a cellule embrionali germinali, le cellule AFS sono state seminate su feeder layer di STO o Sl4-m220, mitoticamente inattivato, con il terreno per le cellule primordiali germinali, supplementato con LIF e il fattore per la crescita dei fibroblasti (bFGF) e successivamente con il terreno N2B27 2iLIF. Per il trattamento dei topi HSA-Cre, SmnF7/F7 le cellule AFS isolate da topi GFP+, sia isolate a fresco che coltivate, sono state iniettate nelle vena della coda e i topi sono stati sacrificati un mese dopo trapianto ed i muscoli analizzati mediante ematossilina ed eosina, tricromica di Masson e con immunofluorescenza per l'espressione di distrofina/GFP. Per studiare l'origine embrionale delle cellule AFS sono stati utilizzati due modelli murini: Oct4-GFP e TNAP-Cre. Per l'induzione alla pluripotenza, cellule ottenute dai feti Oct4-GFP son state trasfettate con i plasmidi del trasposone PB-TET contenente quattro geni (Oct4, Sox2, c-Myc e Klf4) sotto il controllo trascrizionale del promotore inducibile tetO2 tetraciclina/doxiciclina. L'espressione dei geni della pluripotenta è stata indotta con la doxiciclina. Le cellule iPS così ottenute sono state testate per l'espressione dei marcatori Nanog, SSEA-1 e per la positività alla fosfatasi alcalina. Risultati: Le cellule AFS c-kit+ murine variano in numero durante il corso della gestazione. Queste cellule esprimono i marcatori ematopoietici (CD45, CD34, Sca1), mesenchimali (CD90, CD105) insieme a Flk1, CD31, CD44. Sulla base dell'epressione di c-kit sono state identificate due popolazione cellulari: c-kithigh e c-kitlow, che mostrano anche una differente espressione dei marcatori sopracitati. Le cellule c-kitlow sono presenti in numero maggiore e durante il corso della gestazione diminuiscono mentre le c-kithigh aumentano. Entrambe le popolazioni hanno un potenziale ematopoieitco in vitro. Le cellule murine AFS esprimono a bassi livelli i geni Oct4 e Sox2 e ad alti livelli c-Myc e Klf4, ma la loro espressione cambia durante il corso della gestazione. La stessa analisi eseguita a livello di singola cellula ha mostrato che allo stadio E13.5 il 5% delle cellule co-esprime Oct4, Sox2 e Klf4. Le cellule AFS murine non formano teratoma. Negli esperimenti di terapia cellulare topi HSA-Cre, SmnF7/F7 non trattati, morivano all'età di 10 mesi mentre topi trattati con le cellule AFS o cellule da midollo osseo non frazionato avevano una sopravvivvenza del 75% e del 50%, rispettivamente. I topi trattati con le cellule AFS, mostravano un recuperano della forza muscolare rispetto agli animali non trattati (+75%). I muscoli degli animali trattati con le cellule AFS mostravano una morfologia normale con un numero basso di fibre rigeneranti (<1%) e una normale espressione delle distrofina che per il 37.86% (± 9.48%) era GFP+. A 15 mesi dal trattamento, gli animali trattati con cellule del midollo osseo avevano un maggior numero di fibre centro nucleate e una consistente infiltrazione di tessuto interstiziale e nessuna fibra GFP+. Gli animali trattati con le cellule AFS mostravano un fenotipo migliore e il 58.00% (± 2.43%) delle fibre muscolari era GFP+. Risultati simili sono stati ottenuti trattando topi HSA-Cre, SmnF7/F7 con cellule AFS espanse in vitro. Per cercare di differenziare le cellule AFS in cellule embrionali germinali pluripotenti abbiamo testato due protocolli ma nessuna colonia che ricordasse cellule embrionali germinali si è formata in vitro. Le cellule AFS isolate dai feti Oct4-GFP erano GFP negative. Il modello TNAP-Cre è risultato essere aspecifico, perchè TNAP non risultava essere espresso solo nelle cellule germinali primordiali. Negli esperimenti di riprogrammazione, alcuni cloni di iPS hanno mostrato di essere doxiciclina indipendenti, esprimevano il gene Oct4 endogeno ed erano positivi per i marcatori Nanog e SSEA1 e per la fosfatasi alcalina. Discussione: Le cellule murine AFS sono una popolazione eterogenea, le cui caratteristiche variano durante il corso della gestazione ed esprimono marcatori ematopoietici, mesenchimali e anche marcatori tipici delle cellule endoteliali. Il potenziale differenziativo delle due popolazioni c-kithigh e c-kitlow dovrà essere testato in vivo. L'analisi dell'espressione genica a livello di popolazione e a livello di singola cellula per i marcatori della pluripotenza ha confermato l'eterogeneità delle cellule AFS. Il trattamento degli animali HSA-Cre, SmnF7/F7 ha mostrato un potenziale miogenico delle cellule murine AFS, anche a lungo termine, suggerendo un interessante potenziale terapeutico. Le cellule AFS potrebbero contribuire a generare nuove fibre muscolari fondendosi con fibre esistenti o integrarsi nelle nicchia delle cellule staminali muscolari, ma maggiori esperimenti saranno necessari per valutare la validità dell'ipotesi. Anche le cellue AFS coltivate hanno dimostrato il mantenimento delle proprietà rigenerative. Questo studio suggerisce che le cellule AFS murine probabilmente non derivano da cellule primordiali germinali. Tuttavia è importante ricordare che il modello murino Oct4-GFP non è un modello di lineage tracking. Maggiori esperimenti saranno necessari per confermare questi risultati e per identificare l'origine delle cellule AFS. Le cellule iPS sono un promettente strumento di ricerca come modello di malattia o nella speranza di ottenere una sorgente di cellule per terapia. Qui è stato dimostrato come il sistema del PB può essere un valido metodo per la riprogrammazione delle cellule murine AFS. Questi sono solo risultati preliminari e maggiori esperimenti saranno necessari per completarne la caratterizzazione

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects

Necroptosis Is Active in Children With Inflammatory Bowel Disease and Contributes to Heighten Intestinal Inflammation

OBJECTIVES: A new caspase-independent mode of programmed cell death, termed necroptosis, has recently been identified. Altered expression of molecules involved in the necroptosis pathway has been shown to trigger intestinal inflammation. The initiation of necroptosis is principally mediated by the release of receptor interacting protein 3 (RIP3) from suppression by caspase-8. Furthermore, it has been suggested that the mixed lineage kinase domain-like (MLKL) factor is an interacting target of RIP3 in active necroptosis. This study aims at investigating the occurrence of necroptosis in children with inflammatory bowel disease (IBD) and its contribution to human intestinal inflammation. METHODS: Biopsy samples were collected from the ileum and colon of 33 children with Crohn's disease, 30 with ulcerative colitis, and 20 healthy controls. Ten children with allergic colitis (AC) were used as non-IBD comparators. RIP3, caspase-8, and MLKL protein expression levels were evaluated by western blotting. The adenocarcinoma cell line HT29 was used for in vitro experiments. RESULTS: RIP3 and MLKL increased (P<0.01) in inflamed tissues of IBD and AC patients, whereas caspase-8 was reduced. No variations were observed in uninflamed tissues of patients. The relationship between RIP3 increase, active necroptosis, and intestinal inflammation was confirmed by in vitro analyses. CONCLUSIONS: We show for the first time that necroptosis is strongly associated with intestinal inflammation in children with IBD and contributes to strengthen the inflammatory process. We believe that RIP3 and MLKL could represent attractive targets for the management of human IBD

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

none11BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.noneFranzin, Chiara; Piccoli, Martina; Serena, Elena; Bertin, Enrica; Urbani, Luca; Luni, Camilla; Pasqualetto, Valerie; Eaton, Simon; Elvassore, Nicola; De Coppi, Paolo; Pozzobon, Michela*Franzin, Chiara; Piccoli, Martina; Serena, Elena; Bertin, Enrica; Urbani, Luca; Luni, Camilla; Pasqualetto, Valerie; Eaton, Simon; Elvassore, Nicola; De Coppi, Paolo; Pozzobon, Michel

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential