Amniotic fluid stem cells from second and third trimester, comparison and potency for regenerative medicine

Amniotic Fluid Stem Cells (AFSCs) can be isolated from the amniotic fluid after amniocentesis that pregnant women undergo during the second trimester of pregnancy, this population has already been characterised and share common features between embryonic stem cells and adult mesenchymal stem cells. AFSCs are identified by specific markers; this study – as already published – focuses on the CD117 (c-Kit) positive fraction of AFSCs. Those cells are of great interest for regenerative medicine purposes considering their potential of differentiation and the relative constant availability, moreover retrieving prenatal autologous cells can offer new strategies for new-borns with congenital malformations or diseases. This study was intended to explore the possibility of isolating AFSCs at term of pregnancy (so at the third trimester, retrieving the amniotic fluid during delivery). Comparison on phenotype and test of potential for different lineages differentiation has been investigated on cells from both trimesters. These cells have a great potential also because they can be kept in culture and their number significantly increased for successive applications. Hypoxia is also a well-known factor that influence culture of stem cells, cultivating cells at lower oxygen tension has already demonstrated various beneficial effects on other stem cells so it has been decided to try this approach to ameliorate the expansion of AFSCs. In vivo experiments were performed to verify in vitro results on angiogenic potential of AFSCs

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

Amniotic fluid stem cells from second and third trimester, comparison and potency for regenerative medicine

Amniotic Fluid Stem Cells (AFSCs) can be isolated from the amniotic fluid after amniocentesis that pregnant women undergo during the second trimester of pregnancy, this population has already been characterised and share common features between embryonic stem cells and adult mesenchymal stem cells. AFSCs are identified by specific markers; this study – as already published – focuses on the CD117 (c-Kit) positive fraction of AFSCs. Those cells are of great interest for regenerative medicine purposes considering their potential of differentiation and the relative constant availability, moreover retrieving prenatal autologous cells can offer new strategies for new-borns with congenital malformations or diseases. This study was intended to explore the possibility of isolating AFSCs at term of pregnancy (so at the third trimester, retrieving the amniotic fluid during delivery). Comparison on phenotype and test of potential for different lineages differentiation has been investigated on cells from both trimesters. These cells have a great potential also because they can be kept in culture and their number significantly increased for successive applications. Hypoxia is also a well-known factor that influence culture of stem cells, cultivating cells at lower oxygen tension has already demonstrated various beneficial effects on other stem cells so it has been decided to try this approach to ameliorate the expansion of AFSCs. In vivo experiments were performed to verify in vitro results on angiogenic potential of AFSCs.Le cellule staminali del liquido amniotico (AFSCs) possono essere isolate dal liquido amniotico in seguito ad amniocentesi cui le donne incinte si sottopongono durante il secondo trimestre della gravidanza, questa popolazione è già stata caratterizzata e condivide proprietà comuni fra le cellule staminali embrionali e le cellule staminali adulte mesenchimali. Le AFSCs possono essere identificate tramite specifici marcatori; in questo studio – com’è già stato pubblicato – si è puntata l’attenzione sulla frazione positiva per CD117 (c-Kit). Queste cellule sono di grande interesse ai fini della medicina rigenerativa, considerando il loro potenziale di differenziamento e la relativa costante disponibilità, inoltre il recupero di cellule prenatali di origine autologa offre la possibilità di nuove strategie per curare i neonati con malformazioni congenite o altre malattie. In questo studio si è voluto esplorare la possibilità di isolare le AFSCs al termine della gravidanza (quindi al terzo trimestre, recuperando il liquido amniotico durante il parto). Sono stati investigati i fenotipi e le potenzialità differenziative di entrambi i trimestri. Queste cellule hanno anche un grande potenziale poiché possono essere mantenute in cultura e il loro numero significativamente aumentato per successive applicazioni. L’ipossia è un ben conosciuto fattore che influenza la coltura delle cellule staminali; coltivare le cellule in basse tensioni di ossigeno è già stato dimostrato avere un effetto benefico su altre cellule staminali, è stato quindi deciso di provare quest’approccio per migliorare l’espansione delle AFSCs. Sono stati effettuati esperimenti in vivo per verificare il potenziale angiogenico delle AFSCs

Efficiency of antisense-mediated exon skipping in normal and mutated DMD genes

In questo lavoro di tesi si è andati ad indagare l’efficacia di alcuni oligonucleotidi antisenso progettati per escludere alcuni esoni da sequenze “sane”. Le cellule dei pazienti utilizzate in questo lavoro invece presentano molte variazioni a livello di delezioni/mutazioni nel trascritto e si è voluto verificare l’applicabilità degli AO ad un contesto “mutato”. Le cellule dei pazienti sono state derivate da biopsie depositate nella banca di campioni bioptici di Telethon a Milano (in collaborazione con l’istituto Besta di Milano), mentre quelle dei donatori sani dalla banca Telethon di Padova (in collaborazione con la Dott.ssa M. Fanin). Sono stati impiegati oligonucleotidi 2’-O-metil-fosforotioati modificati, sintetizzati in collaborazione con il laboratorio del Dott. S. J. Wilton. Per fare questo si sono trasfettati dei mioblasti di pazienti in coltura per introdurre le molecole nelle cellule e consentire lo skipping. Sono inoltre state effettuate prove di verifica della presenza di distrofina tramite immunofluorescenza

Isolation of c-Kit+ human amniotic fluid stem cells from second trimester

Amniotic fluid-derived stem (AFS) cells have been described as an appealing source of stem cells because of their (1) fetal, non-embryonic origin, (2) easy access during pregnancy overcoming the ethical issues related both to the use of human embryonic cells and to the postnatal tissue biopsy with donor site mor- bidity, and (3) their undemanding ability to be expanded. We and others have demonstrated the broad differentiation potential and here we describe the established protocol we developed to obtain c-Kit+ human AFS cells, starting from second trimester amniocentesis samples

Pancreatic beta-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes

Transfer RNAs (tRNAs) are non-coding RNA molecules essential for protein synthesis. Post-transcriptionally they are heavily modified to improve their function, folding and stability. Intronic polymorphisms in CDKAL1, a tRNA methylthiotransferase, are associated with increased type 2 diabetes risk. Loss-of-function mutations in TRMT10A, a tRNA methyltransferase, are a monogenic cause of early onset diabetes and microcephaly. Here we confirm the role of TRMT10A as a guanosine 9 tRNA methyltransferase, and identify tRNA(Gln) and tRNA(iMeth) as two of its targets. Using RNA interference and induced pluripotent stem cell-derived pancreatic beta-like cells from healthy controls and TRMT10A-deficient patients we demonstrate that TRMT10A deficiency induces oxidative stress and triggers the intrinsic pathway of apoptosis in beta-cells. We show that tRNA guanosine 9 hypomethylation leads to tRNA(Gln) fragmentation and that 5'-tRNA(Gln) fragments mediate TRMT10A deficiency-induced beta-cell death. This study unmasks tRNA hypomethylation and fragmentation as a hitherto unknown mechanism of pancre-atic beta-cell demise relevant to monogenic and polygenic forms of diabetes.Peer reviewe

In depth functional characterization of human induced pluripotent stem cell-derived beta cells in vitro and in vivo

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes

In depth functional characterization of human induced pluripotent stem cell-derived beta cells in vitro and in vivo

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes