89319 - Regenerative Medicine: Unfolding the Nanoworld of Stem Cells Towards a Self-Healing Potential -

Learning outcomes

We plan to provide Students with clear-cut evidence that Regenerative Medicine is not simply a hope for the future, but it is now successfully applied in multiple medical contexts. There is compelling evidence that different regenerative approaches, as those discussed from a molecular point of view by Prof. Ventura, can be remarkably translated into the Orthopaedic fields. Within this context, the issue of obtaining tissue derivatives (i.e. the human adipose tissue) processed to a microfractured tissue encompassing an intact stromal-vascular niche with elements of pericyte/mesenchymal stem cell identity, represents a vivid example of the clinical application of concepts viewing the "niche" as a nanotopography bearing physical and chemical cues essential for unfolding the regenerative potential of niche-enclosed regenerative cells. The finding that these cells are primarily pericytes, the actual source for MSCs in vivo, also involves important clinical outcomes. We will finally discuss the clinical application of a relevant part of the course, that is the use of physical energies to reprogram in situ the tissue resident stem cells to maximize their rescuing potential, both in the absence or presence of cell/tissue transplantation. We expect that Students attending this elective course will become able to use the acquired knowledge to boost their curiosity on the most recent and fascinating breakthrough in stem cell biology. We also expect that, from expanding their “vocabulary” of knowledge the Attendants will have the chance to understand or even perform/being involved in research activities paving the way to novel approaches in (stem) cell reprogramming and tissue regeneration. Accordingly, another expected ability is the chance of participating in or applying to research programs in cell therapy and regenerative medicine at both national and international level

Course contents

Regenerative medicine is an interdisciplinary field that applies engineering and life science principles to regenerate diseased and injured tissues and organs. This course will describe the cellular and molecular mechanisms of tissue renewal and regeneration in humans and will discuss what can be learned from spontaneously regenerating animal models, in particular regarding the role of stem cells differentiation and somatic cell dedifferentiation. Emerging regenerative medicine pre-clinical and clinical approaches, in particular those designed for cardiac repair and orthopaedics applications, will be presented. Progress in understanding the self-healing potential by stimulation of stem cell differentiation and somatic cell dedifferentiation is holding promise for a novel perspective for regenerative medicine. Directions for future regenerative medicine therapies will be discussed. The course is composed of two modules addressing basic and translational research (Prof. D'Uva) and clinical applications (Prof. Zanasi).

Basic and Translational Research (Prof. Gabriele D'Uva)

• Endogenous stem cells in different tissues and their contribution to tissue renewal and regeneration
• Lessons from spontaneously regenerating animal models
• Mammalian cardiac regeneration during fetal and neonatal stages
• Direct cardiogenesis as a novel approach for cardiac regeneration: the role of growth factors, cytokines, extracellular matrix components, mechano-transduction, transcription factors, cell cycle regulators, hormones, cellular energy metabolism, oxygen levels, specific cell populations, and miRNAs

The course will start by describing how fast is cell renewal in different tissue and organs, along with the role and regulatory mechanisms of stem cells. We will describe cutting-edge techniques that have been employed to establish the turnover rate and cellular source of slowing renewing tissues, in particular the brain and the heart. If stem cells are found within these tissues will be elucidated, clarifying major differences in their abundance and localization also in relation to the pre- and post-natal developmental stage. Next, examples of successful and unsuccessful stem cells therapies for regenerative purposes will be discussed in the context of multiple tissues, including blood, cornea, skin, brain and the heart.

The course will also introduce to animal models endowed with spontaneous regenerative ability. For example, some amphibians can regenerate whole limbs, retinas, eye lenses, spinal cords, and tails, as well as upper and lower jaws. The involvement of stem cell differentiation or somatic cell dedifferentiation in these processes will be described.

Then, the course will focus on cardiac regeneration. Indeed, the development of therapeutic strategies for cardiac regeneration following severe cardiac damages is an urgent clinical need. First, we will discuss the discovery of the spontaneous cardiac regenerative ability of adult zebrafish, as well as of mammals during foetal and neonatal stages. Studies that identified cardiomyocyte dedifferentiation and proliferation as the cellular mechanism responsible for these regenerative processes will be presented. Finally, several lessons will focus on the emerging strategy for cardiac regeneration named "DIRECT CARDIOGENESIS", which is based on the stimulation of molecular mechanisms triggering the dedifferentiation and proliferation of endogenous cardiomyocytes for boosting the intrinsic regenerative cardiac ability. We will show the feasibility of this strategy by presenting and critically discussing recent research articles, published in top-tier scientific journals, by showing the ability of multiple factors or conditions to enhance cardiomyocyte proliferation and trigger cardiac regeneration after myocardial infarction in adult mammals.

Clinical Applications (Prof. Stefano Zanasi)

• Background on cartilage defects, and their inherent clinical challenges: the origin of Regenerative Orthopedic Surgery.
• The adipose tissue as a source for regenerative processes.
• The paracrine effect and the exosome nanoworld.
• New generation technologies in human fat processing for regenerative purposes.
• The personal experience.
• Combinatorial use of bone marrow and fat tissue derivatives. The use of nanopatterned scaffolds embedding the tissue mixture.
• Unfolding the regenerative cues of bone marrow and adipose tissue through the reconstruction of tissue nanotopography. Evidence for cartilage regeneration.
• Conclusions: adipose tissue combined with bone marrow for the treatment of coin, complex and contained kissing lesions (OA) of knee, ankle and shoulder, concurrently with realignment procedures, can be considered a safe and highly effective therapeutic option.

The course aims at providing compelling evidence that Regenerative Medicine is becoming a clinical reality and that Regenerative Orthopedic Surgery is one of the fields in which Regenerative Medicine has been first unfolded.

We will show that wide areas of severe cartilage defects due to osteoarthritis of the inferior limb, in particular knee and ankle, can be successfully restored through transplant of human bone marrow combined with defined human fat tissue products.

We will focus on the relevance of yielding fluid fat tissue derivatives containing an intact stromal vascular niche, a stem cell nanotopography including elements of pericyte identity.

We will highlight that mesenchymal stem cells (MSCs), acting as major conductors for tissue regeneration in vivo, reside in the niche in the form pericytes, a cell type that is turned into MSCs upon the intervention of tissue injuries.

We will show that it is now possible to develop nanofabricated scaffolds with tailored-oriented architecture and fiber diameter.

We will provide evidence that the combinatorial use of human bone marrow and fat tissue derivatives, embedded in such scaffolds, yields a significantly more enhanced cartilage regeneration, as compared with the rescuing effects elicited by either bone marrow or a fat derivative alone.

We will show how to document all these clinical outcomes by 1.5T NMR, elastosonography and in randomized histological analyses. These diagnostic tools reveal a higher percentage of hyaline cartilage and rare fibrous tissue compared to the outcome of the single, not combined procedures. On these bases, the combined use of autologous non-expanded tissue products made of whole bone marrow and human white adipose tissue derivatives, can be considered as an autologous/homologous strategy for improving the natural capacity for self-healing in damaged osteo-articular tissues.

Readings/Bibliography

The course utilizes PowerPoint presentations, scientific videos, research articles, and reviews, which will be provided to students through the Virtual Learning Environment platform.

Interested in learning about new regenerative approaches for repairing damaged hearts? Check out our review article in Frontiers in Cardiovascular Medicine (2022), which describes methods for stimulating the proliferation of endogenous cardiac muscle cells.

Teaching methods

The course will consist of frontal lessons using computer-assisted presentations and critical discussions of scientific articles. Additionally, students will be encouraged to identify and explore topics of interest using telematic tools such as PubMed Medline.

Attendance at learning activities is mandatory, with a minimum attendance requirement of 66% of lessons to be eligible for the final exam. Professors may authorize excused absences upon receipt of proper documentation justifying illness or serious reasons. Excused absences will not affect a student's attendance record when determining their minimum attendance requirement.

Assessment methods

Students will undergo separate oral examinations for each module, all held on the same day:

• For the Basic and Translational Research module (taught by Prof. D'Uva), students must deliver a concise presentation (5-10 slides, to be presented in approximately 5 minutes) on a specific scientific article chosen from those covered in the Basic and Translational Research module.
• For the Clinical Applications module (taught by Prof. Zanasi), students will present a brief overview (5-10 slides, to be delivered in around 5 minutes) on a topic of interest within the Clinical Applications module.

In both examinations, the slides should primarily comprise images rather than text. The student will present the slides using a laptop, engaging in critical discussion of the data or topic with the Professor and addressing any further issues arising from the presentation.

At the conclusion of each examination, the Professors will assess the student's performance using a scoring system ranging from 18 to 30, based on the following criteria:

• 18 - 19: Limited preparation on a few topics covered in the course, with analytical skills emerging only with assistance from the teacher, presented in generally correct language.
• 20 - 24: Adequate preparation on a restricted range of topics covered in the course, demonstrating the ability to perform independent analysis mainly on executive matters, expressed in correct language.
• 25 - 29: Thorough preparation on a wide range of topics covered in the course, showcasing the ability to make independent choices, engage in critical analysis, and master specific terminology.
• 30 - 30 cum laude: Comprehensive preparation on all topics addressed in the course, demonstrating the ability to make independent choices, engage in critical analysis and synthesis, possess complete mastery of specific terminology, and excel in argumentation and self-reflection.

The final grade for the Elective Course in Regenerative Medicine will be calculated as the weighted average of the grades obtained in the Basic and Translational Research and Clinical Applications modules, considering the credit units (CFU) assigned to each module.

Teaching tools

All the material will be supplied to the students through the Virtual Learning Environment platform.

Office hours

See the website of Gabriele Matteo D'Uva

See the website of Stefano Zanasi