Journal of Clinical Medicine

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Open AccessEditorial

Preclinical Models: Boosting Synergies for Improved Translation

by Chiara Attanasio and Mara Sangiovanni

J. Clin. Med. 2020, 9(4), 1011; https://doi.org/10.3390/jcm9041011 - 3 Apr 2020

Viewed by 1997

Abstract

The field of preclinical models is a very vast arena, in which finding connections among groups acting in apparently very distant research areas can sometimes prove challenging [...] Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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Research

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21 pages, 4649 KiB

Open AccessArticle

Ontogenetic Pattern Changes of Nucleobindin-2/Nesfatin-1 in the Brain and Intestinal Bulb of the Short Lived African Turquoise Killifish

by Alessia Montesano, Elena De Felice, Adele Leggieri, Antonio Palladino, Carla Lucini, Paola Scocco, Paolo de Girolamo, Mario Baumgart and Livia D’Angelo

J. Clin. Med. 2020, 9(1), 103; https://doi.org/10.3390/jcm9010103 - 31 Dec 2019

Cited by 8 | Viewed by 3494

Abstract

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of [...] Read more.

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost African turquoise killifish Nothobranchius furzeri, a consolidated model organism for aging research. We focused our analysis on brain areas responsible for the regulation of food intake and the rostral intestinal bulb, which is analogous of the mammalian stomach. We hypothesize that in our model, the stomach equivalent structure is the main source of NUCB2 mRNA, displaying higher expression levels than those observed in the brain, mainly during aging. Remarkably, its expression significantly increased in the rostral intestinal bulb compared to the brain, which is likely due to the typical anorexia of aging. When analyzing the pattern of expression, we confirmed the distribution in diencephalic areas involved in food intake regulation at all age stages. Interestingly, in the rostral bulb, NUCB2 mRNA was localized in the lining epithelium of young and old animals, while Nesf-1 immunoreactive cells were distributed in the submucosae. Taken together, our results represent a useful basis for gaining deeper knowledge regarding the mechanisms that regulate food intake during vertebrate aging. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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16 pages, 2854 KiB

Open AccessArticle

Human Red Blood Cells as Oxygen Carriers to Improve Ex-Situ Liver Perfusion in a Rat Model

by Daniele Dondossola, Alessandro Santini, Caterina Lonati, Alberto Zanella, Riccardo Merighi, Luigi Vivona, Michele Battistin, Alessandro Galli, Osvaldo Biancolilli, Marco Maggioni, Stefania Villa and Stefano Gatti

J. Clin. Med. 2019, 8(11), 1918; https://doi.org/10.3390/jcm8111918 - 8 Nov 2019

Cited by 13 | Viewed by 3179

Abstract

Ex-situ machine perfusion (MP) has been increasingly used to enhance liver quality in different settings. Small animal models can help to implement this procedure. As most normothermic MP (NMP) models employ sub-physiological levels of oxygen delivery (DO₂), the aim of this [...] Read more.

Ex-situ machine perfusion (MP) has been increasingly used to enhance liver quality in different settings. Small animal models can help to implement this procedure. As most normothermic MP (NMP) models employ sub-physiological levels of oxygen delivery (DO₂), the aim of this study was to investigate the effectiveness and safety of different DO₂, using human red blood cells (RBCs) as oxygen carriers on metabolic recovery in a rat model of NMP. Four experimental groups (n = 5 each) consisted of (1) native (untreated/control), (2) liver static cold storage (SCS) 30 min without NMP, (3) SCS followed by 120 min of NMP with Dulbecco-Modified-Eagle-Medium as perfusate (DMEM), and (4) similar to group 3, but perfusion fluid was added with human RBCs (hematocrit 15%) (BLOOD). Compared to DMEM, the BLOOD group showed increased liver DO₂ (p = 0.008) and oxygen consumption (

\dot{V O}

₂) (p < 0.001); lactate clearance (p < 0.001), potassium (p < 0.001), and glucose (p = 0.029) uptake were enhanced. ATP levels were likewise higher in BLOOD relative to DMEM (p = 0.031).

\dot{V O}

₂ and DO₂ were highly correlated (p < 0.001). Consistently, the main metabolic parameters were directly correlated with DO₂ and

\dot{V O}

₂. No human RBC related damage was detected. In conclusion, an optimized DO₂ significantly reduces hypoxic damage-related effects occurring during NMP. Human RBCs can be safely used as oxygen carriers. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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13 pages, 4416 KiB

Open AccessArticle

3D Bioprinted Human Cortical Neural Constructs Derived from Induced Pluripotent Stem Cells

by Federico Salaris, Cristina Colosi, Carlo Brighi, Alessandro Soloperto, Valeria de Turris, Maria Cristina Benedetti, Silvia Ghirga, Maria Rosito, Silvia Di Angelantonio and Alessandro Rosa

J. Clin. Med. 2019, 8(10), 1595; https://doi.org/10.3390/jcm8101595 - 2 Oct 2019

Cited by 47 | Viewed by 6083

Abstract

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent [...] Read more.

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent stem cells (iPSCs). Human iPSCs can be differentiated in a wide range of neurons and glia, providing an ideal tool for modeling the human nervous system. Here we report a neural construct generated by 3D bioprinting of cortical neurons and glial precursors derived from human iPSCs. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. This work lays the basis for generating more complex and faithful 3D models of the human nervous systems by bioprinting neural cells derived from iPSCs. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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16 pages, 9841 KiB

Open AccessArticle

Identification and Expression of Neurotrophin-6 in the Brain of Nothobranchius furzeri: One More Piece in Neurotrophin Research

by Adele Leggieri, Chiara Attanasio, Antonio Palladino, Alessandro Cellerino, Carla Lucini, Marina Paolucci, Eva Terzibasi Tozzini, Paolo de Girolamo and Livia D’Angelo

J. Clin. Med. 2019, 8(5), 595; https://doi.org/10.3390/jcm8050595 - 30 Apr 2019

Cited by 12 | Viewed by 3466

Abstract

Neurotrophins contribute to the complexity of vertebrate nervous system, being involved in cognition and memory. Abnormalities associated with neurotrophin synthesis may lead to neuropathies, neurodegenerative disorders and age-associated cognitive decline. The genome of teleost fishes contains homologs of some mammalian neurotrophins as well [...] Read more.

Neurotrophins contribute to the complexity of vertebrate nervous system, being involved in cognition and memory. Abnormalities associated with neurotrophin synthesis may lead to neuropathies, neurodegenerative disorders and age-associated cognitive decline. The genome of teleost fishes contains homologs of some mammalian neurotrophins as well as a gene coding for an additional neurotrophin (NT-6). In this study, we characterized this specific neurotrophin in the short-lived fish Nothobranchius furzeri, a relatively new model for aging studies. Thus, we report herein for the first time the age-related expression of a neurotrophin in a non-mammalian vertebrate. Interestingly, we found comparable expression levels of NT-6 in the brain of both young and old animals. More in detail, we used a locked nucleic acid probe and a riboprobe to investigate the neuroanatomical distribution of NT-6 mRNA revealing a significant expression of the neurotrophin in neurons of the forebrain (olfactory bulbs, dorsal and ventral telencephalon, and several diencephalic nuclei), midbrain (optic tectum, longitudinal tori, and semicircular tori), and hindbrain (valvula and body of cerebellum, reticular formation and octavolateral area of medulla oblongata). By combining in situ hybridization and immunohistochemistry, we showed that NT-6 mRNA is synthesized in mature neurons. These results contribute to better understanding the evolutionary history of neurotrophins in vertebrates, and their role in the adult brain. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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19 pages, 4443 KiB

Open AccessArticle

New Chondrosarcoma Cell Lines with Preserved Stem Cell Properties to Study the Genomic Drift During In Vitro/In Vivo Growth

by Veronica Rey, Sofia T. Menendez, Oscar Estupiñan, Aida Rodriguez, Laura Santos, Juan Tornin, Lucia Martinez-Cruzado, David Castillo, Gonzalo R. Ordoñez, Serafin Costilla, Carlos Alvarez-Fernandez, Aurora Astudillo, Alejandro Braña and Rene Rodriguez

J. Clin. Med. 2019, 8(4), 455; https://doi.org/10.3390/jcm8040455 - 4 Apr 2019

Cited by 22 | Viewed by 3833

Abstract

For the cancer genomics era, there is a need for clinically annotated close-to-patient cell lines suitable to investigate altered pathways and serve as high-throughput drug-screening platforms. This is particularly important for drug-resistant tumors like chondrosarcoma which has few models available. Here we established [...] Read more.

For the cancer genomics era, there is a need for clinically annotated close-to-patient cell lines suitable to investigate altered pathways and serve as high-throughput drug-screening platforms. This is particularly important for drug-resistant tumors like chondrosarcoma which has few models available. Here we established and characterized new cell lines derived from two secondary (CDS06 and CDS11) and one dedifferentiated (CDS-17) chondrosarcomas as well as another line derived from a CDS-17-generated xenograft (T-CDS17). These lines displayed cancer stem cell-related and invasive features and were able to initiate subcutaneous and/or orthotopic animal models. Different mutations in Isocitrate Dehydrogenase-1 (IDH1), Isocitrate Dehydrogenase-2 (IDH2), and Tumor Supressor P53 (TP53) and deletion of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) were detected both in cell lines and tumor samples. In addition, other mutations in TP53 and the amplification of Mouse Double Minute 2 homolog (MDM2) arose during cell culture in CDS17 cells. Whole exome sequencing analysis of CDS17, T-CDS17, and matched patient samples confirmed that cell lines kept the most relevant mutations of the tumor, uncovered new mutations and revealed structural variants that emerged during in vitro/in vivo growth. Altogether, this work expanded the panel of clinically and genetically-annotated chondrosarcoma lines amenable for in vivo studies and cancer stem cell (CSC) characterization. Moreover, it provided clues of the genetic drift of chondrosarcoma cells during the adaptation to grow conditions. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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Review

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21 pages, 1392 KiB

Open AccessReview

Radiolabeled PET/MRI Nanoparticles for Tumor Imaging

by Ernesto Forte, Dario Fiorenza, Enza Torino, Angela Costagliola di Polidoro, Carlo Cavaliere, Paolo A. Netti, Marco Salvatore and Marco Aiello

J. Clin. Med. 2020, 9(1), 89; https://doi.org/10.3390/jcm9010089 - 29 Dec 2019

Cited by 55 | Viewed by 6715

Abstract

The development of integrated positron emission tomography (PET)/magnetic resonance imaging (MRI) scanners opened a new scenario for cancer diagnosis, treatment, and follow-up. Multimodal imaging combines functional and morphological information from different modalities, which, singularly, cannot provide a comprehensive pathophysiological overview. Molecular imaging exploits [...] Read more.

The development of integrated positron emission tomography (PET)/magnetic resonance imaging (MRI) scanners opened a new scenario for cancer diagnosis, treatment, and follow-up. Multimodal imaging combines functional and morphological information from different modalities, which, singularly, cannot provide a comprehensive pathophysiological overview. Molecular imaging exploits multimodal imaging in order to obtain information at a biological and cellular level; in this way, it is possible to track biological pathways and discover many typical tumoral features. In this context, nanoparticle-based contrast agents (CAs) can improve probe biocompatibility and biodistribution, prolonging blood half-life to achieve specific target accumulation and non-toxicity. In addition, CAs can be simultaneously delivered with drugs or, in general, therapeutic agents gathering a dual diagnostic and therapeutic effect in order to perform cancer diagnosis and treatment simultaneous. The way for personalized medicine is not so far. Herein, we report principles, characteristics, applications, and concerns of nanoparticle (NP)-based PET/MRI CAs. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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36 pages, 1734 KiB

Open AccessReview

The Reeler Mouse: A Translational Model of Human Neurological Conditions, or Simply a Good Tool for Better Understanding Neurodevelopment?

by Laura Lossi, Claudia Castagna, Alberto Granato and Adalberto Merighi

J. Clin. Med. 2019, 8(12), 2088; https://doi.org/10.3390/jcm8122088 - 1 Dec 2019

Cited by 21 | Viewed by 7699

Abstract

The first description of the Reeler mutation in mouse dates to more than fifty years ago, and later, its causative gene (reln) was discovered in mouse, and its human orthologue (RELN) was demonstrated to be causative of lissencephaly 2 [...] Read more.

The first description of the Reeler mutation in mouse dates to more than fifty years ago, and later, its causative gene (reln) was discovered in mouse, and its human orthologue (RELN) was demonstrated to be causative of lissencephaly 2 (LIS2) and about 20% of the cases of autosomal-dominant lateral temporal epilepsy (ADLTE). In both human and mice, the gene encodes for a glycoprotein referred to as reelin (Reln) that plays a primary function in neuronal migration during development and synaptic stabilization in adulthood. Besides LIS2 and ADLTE, RELN and/or other genes coding for the proteins of the Reln intracellular cascade have been associated substantially to other conditions such as spinocerebellar ataxia type 7 and 37, VLDLR-associated cerebellar hypoplasia, PAFAH1B1-associated lissencephaly, autism, and schizophrenia. According to their modalities of inheritances and with significant differences among each other, these neuropsychiatric disorders can be modeled in the homozygous (reln^−/−) or heterozygous (reln^+/−) Reeler mouse. The worth of these mice as translational models is discussed, with focus on their construct and face validity. Description of face validity, i.e., the resemblance of phenotypes between the two species, centers onto the histological, neurochemical, and functional observations in the cerebral cortex, hippocampus, and cerebellum of Reeler mice and their human counterparts. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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27 pages, 2074 KiB

Open AccessReview

Bioengineered Skin Substitutes: The Role of Extracellular Matrix and Vascularization in the Healing of Deep Wounds

by Francesco Urciuolo, Costantino Casale, Giorgia Imparato and Paolo A. Netti

J. Clin. Med. 2019, 8(12), 2083; https://doi.org/10.3390/jcm8122083 - 1 Dec 2019

Cited by 64 | Viewed by 10825

Abstract

The formation of severe scars still represents the result of the closure process of extended and deep skin wounds. To address this issue, different bioengineered skin substitutes have been developed but a general consensus regarding their effectiveness has not been achieved yet. It [...] Read more.

The formation of severe scars still represents the result of the closure process of extended and deep skin wounds. To address this issue, different bioengineered skin substitutes have been developed but a general consensus regarding their effectiveness has not been achieved yet. It will be shown that bioengineered skin substitutes, although representing a valid alternative to autografting, induce skin cells in repairing the wound rather than guiding a regeneration process. Repaired skin differs from regenerated skin, showing high contracture, loss of sensitivity, impaired pigmentation and absence of cutaneous adnexa (i.e., hair follicles and sweat glands). This leads to significant mobility and aesthetic concerns, making the development of more effective bioengineered skin models a current need. The objective of this review is to determine the limitations of either commercially available or investigational bioengineered skin substitutes and how advanced skin tissue engineering strategies can be improved in order to completely restore skin functions after severe wounds. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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29 pages, 332 KiB

Open AccessReview

Magnetic Resonance Imaging for Translational Research in Oncology

by Maria Felicia Fiordelisi, Carlo Cavaliere, Luigi Auletta, Luca Basso and Marco Salvatore

J. Clin. Med. 2019, 8(11), 1883; https://doi.org/10.3390/jcm8111883 - 6 Nov 2019

Cited by 9 | Viewed by 3422

Abstract

The translation of results from the preclinical to the clinical setting is often anything other than straightforward. Indeed, ideas and even very intriguing results obtained at all levels of preclinical research, i.e., in vitro, on animal models, or even in clinical trials, often [...] Read more.

The translation of results from the preclinical to the clinical setting is often anything other than straightforward. Indeed, ideas and even very intriguing results obtained at all levels of preclinical research, i.e., in vitro, on animal models, or even in clinical trials, often require much effort to validate, and sometimes, even useful data are lost or are demonstrated to be inapplicable in the clinic. In vivo, small-animal, preclinical imaging uses almost the same technologies in terms of hardware and software settings as for human patients, and hence, might result in a more rapid translation. In this perspective, magnetic resonance imaging might be the most translatable technique, since only in rare cases does it require the use of contrast agents, and when not, sequences developed in the lab can be readily applied to patients, thanks to their non-invasiveness. The wide range of sequences can give much useful information on the anatomy and pathophysiology of oncologic lesions in different body districts. This review aims to underline the versatility of this imaging technique and its various approaches, reporting the latest preclinical studies on thyroid, breast, and prostate cancers, both on small laboratory animals and on human patients, according to our previous and ongoing research lines. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

25 pages, 2354 KiB

Open AccessReview

Modular Strategies to Build Cell-Free and Cell-Laden Scaffolds towards Bioengineered Tissues and Organs

by Aurelio Salerno, Giuseppe Cesarelli, Parisa Pedram and Paolo Antonio Netti

J. Clin. Med. 2019, 8(11), 1816; https://doi.org/10.3390/jcm8111816 - 1 Nov 2019

Cited by 28 | Viewed by 4914

Abstract

Engineering three-dimensional (3D) scaffolds for functional tissue and organ regeneration is a major challenge of the tissue engineering (TE) community. Great progress has been made in developing scaffolds to support cells in 3D, and to date, several implantable scaffolds are available for treating [...] Read more.

Engineering three-dimensional (3D) scaffolds for functional tissue and organ regeneration is a major challenge of the tissue engineering (TE) community. Great progress has been made in developing scaffolds to support cells in 3D, and to date, several implantable scaffolds are available for treating damaged and dysfunctional tissues, such as bone, osteochondral, cardiac and nerve. However, recapitulating the complex extracellular matrix (ECM) functions of native tissues is far from being achieved in synthetic scaffolds. Modular TE is an intriguing approach that aims to design and fabricate ECM-mimicking scaffolds by the bottom-up assembly of building blocks with specific composition, morphology and structural properties. This review provides an overview of the main strategies to build synthetic TE scaffolds through bioactive modules assembly and classifies them into two distinct schemes based on microparticles (µPs) or patterned layers. The µPs-based processes section starts describing novel techniques for creating polymeric µPs with desired composition, morphology, size and shape. Later, the discussion focuses on µPs-based scaffolds design principles and processes. In particular, starting from random µPs assembly, we will move to advanced µPs structuring processes, focusing our attention on technological and engineering aspects related to cell-free and cell-laden strategies. The second part of this review article illustrates layer-by-layer modular scaffolds fabrication based on discontinuous, where layers’ fabrication and assembly are split, and continuous processes. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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18 pages, 1656 KiB

Open AccessReview

Induced Pluripotent Stem Cells as Vasculature Forming Entities

by Antonio Palladino, Isabella Mavaro, Carmela Pizzoleo, Elena De Felice, Carla Lucini, Paolo de Girolamo, Paolo A. Netti and Chiara Attanasio

J. Clin. Med. 2019, 8(11), 1782; https://doi.org/10.3390/jcm8111782 - 25 Oct 2019

Cited by 10 | Viewed by 4120

Abstract

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to [...] Read more.

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine. Full article

(This article belongs to the Special Issue Cutting Edge Preclinical Models in Translational Medicine)

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