the-production-pluripotent-stem-cells-from-mouse-amniotic-fluid-cells

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 ...

Fondazione Istituto di Ricerca Pediatrica Citta della Speranza

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCbeta activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCbeta siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

High glucose induces adipogenic differentiation of muscle-derived stem cells.

Wnt signaling inhibits adipogenesis, but its regulation, physiological relevance, and molecular effectors are poorly understood. Here, we identify the Wnt modulator Dapper1/Frodo1 (Dact1) as a new preadipocyte gene involved in the regulation of murine and human adipogenesis.

Dact1, a nutritionally regulated preadipocyte gene, controls adipogenesis by coordinating the Wnt/beta-catenin signaling network.

The intermuscular adipose tissue (IMAT) is a depot of adipocytes located between muscle bundles. Several investigations have recently been carried out to define the phenotype, the functional characteristics, and the origin of the adipocytes present in this depot. Among the different mechanisms that could be responsible for the accumulation of fat in this site, the dysdifferentiation of muscle-derived stem cells or other mesenchymal progenitors has been postulated, turning them into cells with an adipocyte phenotype. In particular, muscle satellite cells (SCs), a heterogeneous stem cell population characterized by plasticity and self-renewal that allow muscular growth and regeneration, can acquire features of adipocytes, including the abilities to express adipocyte-specific genes and accumulate lipids. Failure to express the transcription factors that direct mesenchymal precursors into fully differentiated functionally specialized cells may be responsible for their phenotypic switch into the adipogenic lineage. We proved that human SCs also possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle. This occurs under some pathological conditions (i.e., primary myodystrophies, obesity, hyperglycemia, high plasma free fatty acids, hypoxia, etc.) or as a consequence of thiazolidinedione treatment or simply because of a sedentary lifestyle or during aging. Several pathways and factors (PPARs, WNT growth factors, myokines, GEF-GAP-Rho, p66(shc), mitochondrial ROS production, PKCÎ²) could be implicated in the adipogenic conversion of SCs. The understanding of the molecular pathways that regulate muscle-to-fat conversion and SC behavior could explain the increase in IMAT depots that characterize many metabolic diseases and age-related sarcopenia.

The origin of intermuscular adipose tissue and its pathophysiological implications.

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (approximately 75%) and the high proliferative clones (HPC), present instead in minor amount (approximately 25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (DeltaPsi(m)), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

In this study we investigated the effect of human amniotic fluid stem (hAFS) cells and rat adipose tissue stromal vascular fraction GFP-positive cell (rSVC-GFP) therapy and the contribution of the paracrine and neurohormonal milieu to cardiac and pulmonary vascular remodeling in a rat model of pulmonary hypertension (PH) and right heart failure (RHF).

Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: role of paracrine and neurohormonal milieu in the remodeling process.

Mutations in the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophy, a fatal neuromuscular disorder. Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted. Herein we demonstrate that tail vein transplantation of mouse amniotic fluid stem (AFS) cells enhances the muscle strength and improves the survival rate of the affected animals. Second, after cardiotoxin injury of the Tibialis Anterior, only AFS-transplanted mice efficiently regenerate. Most importantly, secondary transplants of satellite cells (SCs) derived from treated mice show that AFS cells integrate into the muscle stem cell compartment and have long-term muscle regeneration capacity indistinguishable from that of wild-type-derived SC. This is the first study demonstrating the functional and stable integration of AFS cells into the skeletal muscle, highlighting their value as cell source for the treatment of muscular dystrophies.

Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model.

Satellite cells (SCs) are essential for postnatal muscle growth and regeneration, however, their expansion potential in vitro is limited. Recently, hypoxia has been used to enhance proliferative abilities in vitro of various primary cultures. Here, by isolating SCs from single mouse hindlimb skeletal myofibers, we were able to distinguish two subpopulations of clonally cultured SCs (Low Proliferative Clones--LPC--and High Proliferative Clones--HPC), which, as shown in rat skeletal muscle, were present at a fixed proportion. In addition, culturing LPC and HPC at a low level of oxygen we observed a two fold increased proliferation both for LPC and HPC. LPC showed higher myogenic regulatory factor (MRF) expression than HPC, particularly under the hypoxic condition. Notably, a different myogenic potential between LPC and HPC was retained in vivo: green fluorescent protein (GFP)+LPC transplantation in cardiotoxin-injured Tibialis Anterior led to a higher number of new GFP+muscle fibers per transplanted cell than GFP+HPC. Interestingly, the in vivo myogenic potential of a single cell from an LPC is similar if cultured both in normoxia and hypoxia. Therefore, starting from a single satellite cell, hypoxia allows a larger expansion of LPC than normal O(2) conditions, obtaining a consistent amount of cells for transplantation, but maintaining their myogenic regeneration potential.

Hypoxia increases mouse satellite cell clone proliferation maintaining both in vitro and in vivo heterogeneity and myogenic potential.

BACKGROUND: The aim of our study was to investigate whether stem cell (SC) therapy with human amniotic fluid stem cells (hAFS, fetal stem cells) and rat adipose tissue stromal vascular fraction cells-GFP positive cells (rSVC-GFP) was able to produce favorable effects on skeletal muscle (SM) remodeling in a well-established rat model of right heart failure (RHF). METHODS: RHF was induced by monocrotaline (MCT) in Sprague-Dawley rats. Three weeks later, four millions of hAFS or rSVC-GFP cells were injected via tail vein. SM remodeling was assessed by Soleus muscle fiber cross sectional area (CSA), myocyte apoptosis, myosin heavy chain (MHC) composition, satellite cells pattern, and SC immunohistochemistry. RESULTS: hAFS and rSVC-GFP injection produced significant SC homing in Soleus (0.68Â±1.0 and 0.67Â±0.75% respectively), with a 50% differentiation toward smooth muscle and endothelial cells. Pro-inflammatory cytokines were down regulated to levels similar to those of controls. SC-treated (SCT) rats showed increased CSA (p<0.004 vs MCT) similarly to controls with a reshift toward the slow MHC1 isoform. Apoptosis was significantly decreased (11.12.Â±8.8cells/mm(3) hAFS and 13.1+7.6 rSVC-GFP) (p<0.001 vs MCT) and similar to controls (5.38Â±3.0cells/mm(3)). RHF rats showed a dramatic reduction of satellite cells(MCT 0.2Â±0.06% Pax7 native vs controls 2.60Â±2.46%, p<0.001), while SCT induced a repopulation of both native and SC derived satellite cells (p<0.005). CONCLUSIONS: SC treatment led to SM remodeling with satellite cell repopulation, decreased atrophy and apoptosis. Modulation of the cytokine milieu might play a crucial pathophysiological role with a possible scenario for autologous transplantation of SC in pts with CHF myopathy.

The contribution of stem cell therapy to skeletal muscle remodeling in heart failure.

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 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.

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

More than 40% of the body mass is represented by muscle tissue, which possesses the innate ability to regenerate after damage through the activation of muscle-specific stem cells, namely satellite cells. Muscle diseases, in particular chronic degenerative states of skeletal muscle such as dystrophies, lead to a perturbation of the regenerative process, which causes the premature exhaustion of satellite cell reservoir due to continuous cycles of degeneration/regeneration. Nowadays, the research is focused on different therapeutic approaches, ranging from gene and cell to pharmacological therapy, but still there is no definitive cure in particular for genetic muscle disease. Keeping this in mind, in this article, we will give special consideration to muscle diseases and the use of fetal derived stem cells as a new approach for therapy. Cells of fetal origin, from cord blood to placenta and amniotic fluid, can be easily obtained without ethical concern, expanded and differentiated in culture, and possess immune-modulatory properties. The in vivo approach in animal models can be helpful to study the mechanism underneath the operating principle of the stem cell reservoir, namely the niche, which holds great potential to understand the onset of muscle pathologies.

Fetal stem cells and skeletal muscle regeneration: a therapeutic approach.

Endothelial dysfunction is found in different pathologies such as diabetes and renal and heart diseases, representing one of the major health problems. The reduced vasodilation of impaired endothelium starts a prothrombotic state associated with irregular blood flow. We aimed to explore the potential of amniotic fluid stem (AFS) cells as a source for regenerative medicine in this field; for the first time, we focused on third-trimester amniotic fluid AFS cells and compared them with the already-described AFS cells from the second trimester.

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

One of the major issues concerning human skeletal muscle progenitor cells is represented by the efficient isolation and in vitro expansion of cells retaining the ability to proliferate, migrate and differentiate once transplanted. Here we describe a method (1) effective in obtaining human muscle precursor cells both from fresh and frozen biopsies coming from different muscles, (2) selective to yield cells uniformly positive for CD56 and negative for CD34 without FACS sorting, (3) reliable in maintaining proliferative and in vitro differentiative capacity up to passage 10.

Isolation and Expansion of Muscle Precursor Cells from Human Skeletal Muscle Biopsies.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EV(Normo) and EV(Hypo) ). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EV(Normo) and EV(Hypo) interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206(pos) cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EV(Hypo) administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. © Stem Cells Translational Medicine 2017.

Article	Year
High glucose induces adipogenic differentiation of muscle-derived stem cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18212116	2008
Dact1, a nutritionally regulated preadipocyte gene, controls adipogenesis by coordinating the Wnt/beta-catenin signaling network. Diabetes\| PubMed ID: 19073771	2009
The origin of intermuscular adipose tissue and its pathophysiological implications. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 19738037	2009
Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity. PloS one\| PubMed ID: 20049087	2010
Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: role of paracrine and neurohormonal milieu in the remodeling process. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation\| PubMed ID: 21989772	2011
Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model. Stem cells (Dayton, Ohio)\| PubMed ID: 22644669	2012
Hypoxia increases mouse satellite cell clone proliferation maintaining both in vitro and in vivo heterogeneity and myogenic potential. PloS one\| PubMed ID: 23166781	2012
The contribution of stem cell therapy to skeletal muscle remodeling in heart failure. International journal of cardiology\| PubMed ID: 23453873	2013
Single cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers. Biology of the cell / under the auspices of the European Cell Biology Organization\| PubMed ID: 24024612	2013
Fetal stem cells and skeletal muscle regeneration: a therapeutic approach. Frontiers in aging neuroscience\| PubMed ID: 25221507	2014
Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia. Stem cell research & therapy\| PubMed ID: 26519360	2015
Isolation and Expansion of Muscle Precursor Cells from Human Skeletal Muscle Biopsies. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27032940	2016
Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization. Stem cells translational medicine\| PubMed ID: 28186708	2017

Chiara Franzin

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