Pubblicazioni scientifiche

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Pubblicazioni scientifiche

RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole genome sequencing

Author

Falzarano MS, Grilli A, Zia S, Fang M, Rossi R,  Gualandi F, Rimessi P, Dani RE, Fabris M, Lu Z, Li W, Mongini T, Ricci F, Pegoraro E, Bello L, Barp A, Sansone VA, Hegde M, Roda B, Reschiglian P, Bicciato S, Selvatici R, Ferlini A

Publication

HGG Advances, Available online 24 August 2021

Abstract

Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification.

Gene expression profiling of DMD patients’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirror the known transcriptional landscape of DMD muscle and worsen following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy.

The full DMD transcript profile performed in USCs from three undiagnosed DMD patients addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole genome sequencing. This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions respectively. All three mutations affect DMD gene splicing, cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector®) highlighted cell-type variability and the representation of cell-specific DMD isoforms.

Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

A New Predictive Technology for Perinatal Stem Cell Isolation Suited for Cell Therapy Approaches

Author

Zia S, Martini G, Pizzuti V, Maggio A, Simonazzi G, Reschiglian P, Bonsi L, Alviano F, Roda B and Zattoni A

Publication

Micromachines 2021, 12, 782

Abstract

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate; while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations

Author

Casciaro F, Zia S, Forcato M, Zavatti M, Beretti F, Bertucci E, Zattoni A, Reschiglian P, Alviano F, Bonsi L, Follo MY, Demaria M, Roda B, Maraldi T

Publication

Cells, 2021 Jan 15;10(1):158.

Abstract

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment

Authors
Rossi M, Roda B, Zia S, Vigliotta I, Zannini C, Alviano F, Bonsi L, Zattoni A, Reschiglian P, Gennai A

Publication
Aesthetic Surgery Journal, published online 2018 Jun 14

Abstract

Background
New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered.

Objectives
To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples.

Methods
Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays.

Results
Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages.

Conclusions
Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Recent patents and advances on tag-less microfluidic stem cell sorting methods: Applications for perinatal stem cell isolation

Authors
Alviano F, Roda B, Rossi, M, Tanase M, Martinelli K, Marchionni C, Zattoni, Reschiglian P,  Bonsi L

Publication
Recent Patents on Regenerative Medicine 3 (2013) 215-226

Abstract

Interest in stem cell separation and purification from easily accessible clinical specimens is booming due to the increase of cell therapy applications. The recovery of pluripotent or multipotent stem cells in human sources different from the embryo requires the use of effective methods of cell sorting/enrichment. Among these sources, perinatal tissues retain cells with pivotal stem cell features such as high self-renewal ability, wide differentiation potential and immunomodulatory properties. In this perspective, methods exploiting cell biophysical differences in a less dependent process of identification based on specific markers are therefore promising. These methods allow cell isolation irrespective of the broad and diversified surface antigenic panel that usually limits the ability to easily distinguish cells as in the case of mesenchymal stromal/stem cell separation. In addition, the use of non- or minimally invasive tag-less techniques might be a way to preserve stem cell features of the selected product and reduce regulatory issues related to their use in regenerative applications. In this review, non-invasive cell sorting techniques based on microfluidic systems and relevant patents are described. In particular applications of emerging separation approach, Field-Flow Fractionation (FFF), for perinatal stem cell sorting are cited. Protocols and applications based on FFF-derived techniques are detailed.

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation

Authors
Lattuada D, Roda B, Pignatari C, Magni R, Colombo F, Cattaneo A, Zattoni A, Cetin I, Reschiglian P, Bolis G

Publication
Analytical and Bioanalytical Chemistry 405 (2013) 977-84

Abstract

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

A novel stem cell tag-less sorting method

Authors
Roda B, Lanzoni G, Alviano F, Zattoni A, Costa R, Di Carlo A, Marchionni C, Franchina M, Ricci F, Tazzari PL, Pagliaro P, Scalinci SZ, Bonsi L, Reschiglian P, Bagnara GP

Publication
Stem Cell Reviews 5 (2009) 420-7

Abstract

Growing interest in stem cell research has led to the development of a number of new methods for isolation. The lack of homogeneity in stem cell preparation blurs standardization, which however is recommended for successful applications. Among stem cells, mesenchymal stem cells (MSCs) are promising candidates for cell therapy applications. This paper presents a fractionation protocol based on a tag-less, flow-assisted method of purifying, distinguishing and sorting MSCs. The protocol entails a suspension of cells in a transport fluid being injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min) sorted cells are collected. The protocol has been applied to the improvement of MSC isolation, with a specific view to reducing cell manipulation operations, keeping instrumental simplicity and increasing analytical information for cell characterization. Applications such as MSC purification from epithelial contaminants, MSC characterization from various human sources and sorting of MSC subpopulations with high differentiation potential are described. The low cost, full biocompatibility and scale-up potential of the protocol presented could make the procedure attractive for stem cell selection.

A tag-less method of sorting stem cells from clinical specimens and separating mesenchymal from epithelial progenitor cells

Authors
Roda B, Reschiglian P, Zattoni A, Alviano F, Lanzoni G, Costa R, Di Carlo A, Marchionni C, Franchina M, Bonsi L, Bagnara GP

Publication
Cytometry B - Clinical Cytometry 76 (2009) 285-90

Abstract

Background
The interest in stem cell (SC) isolation from easily accessible clinical specimens is booming. The lack of homogeneity in pluri/multipotent SC preparation blurs standardization, which however is recommended for successful applications. Multipotent mesenchymal SCs (MSCs) in fact express a broad panel of surface antigens, which limit the possibility of sorting homogeneous preparations by using an immunotag‐based method.

 

Methods
We present a tag‐less, flow‐assisted method to purify, distinguish, and sort pluri/multipotent SCs obtained from clinical specimens, based on differences in the biophysical properties that cells acquire when in suspension under fluidic conditions. A suspension of cells in a transport fluid is injected into a ribbon‐like capillary device by continuous flow. In a relatively short time (about 30 min), sorted cells are collected.

 

Results
We obtained baseline separation between MSCs and epithelial cells, which are important contaminants of isolated MSCs. The extent of separation is evaluated by flow cytometry through detection of a specific epithelial antigen. MSCs from various human sources also prove to have different, characteristic, highly‐reproducible fractionation profiles. Finally, we evaluated the dissimilar differentiation potential among cell fractions obtained from sorting a single MSC source. After differentiation induction, a fraction displayed a differentiation yield close to 100%, whereas unfractionated cells contained only 40% of responding cells.

 

Conclusions
The results demonstrate that the method presented is able to obtain selected and well‐characterized living MSCs with an increased differentiation yield. Its reduced cost, full biocompatibility, and scale‐up potential could make this method an effective procedure for stem cell selection.

Human lymphocyte sorting by gravitational field-flow fractionation

Authors
Roda B, Reschiglian P, Zattoni A, Tazzari PL, Buzzi M, Ricci F, Bontadini A

Publication
Analytical and Bioanalytical Chemistry 392 (2008) 137-45

Abstract

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.

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