Ebook: Stem Cells and Regenerative Medicine

Most human tissues do not regenerate spontaneously. This is why the development of biotherapies with stem cells represents promising alternatives. The annual expenses linked to these innovative treatments is estimated to more than 100 billion euros over the next 10 years.

Among possible medium-term therapeutic applications are, cardiac insufficiency, preparation of small diameter arteries, treatment of atherosclerosis, bone repair, cartilage defects, burns, diabetes, liver or bladder regeneration, and neurodegenerative disorders. This concept of regenerative medicine is an emerging multidisciplinary field involving surgery medicine, biology, chemistry, mechanics and engineering that can be defined as «the way to improve the health and quality of life by restoring, maintaining, or enhancing tissue and organ functions».

Cell therapy and regenerative medicine are taking a growing place as innovative treatments for the replacement or repair of aged or dead cells or tissues. Indeed many organs are progressively and irreversibly damaged during aging, or after severe degenerative diseases, thus finally affecting their function. This process leads to an alteration of the patient’s life quality, and often threats its survival. Regenerative medicine, as a new therapeutic strategy, aims at restoring defective organs by several biological tools or processes, especially the use of mesenchymal stem cells. Regenerative medicine relies on multi-disciplinary approaches of cell and tissue engineering at the cross road of biological and engineering sciences involving a close partnership between biologists, physico-chemists, biomechanics, clinicians, thus providing a continuum from basic science to the clinics. One of the best synergy examples between cell biology and engineering is the extended use of the 3D-bioprinting which helps in the efficient building of organs such as liver or cartilage.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate in different tissues. Although, autologous cells were previously chosen as the best candidates, these cells are generally not easily available. They are frequently in a pathological state and expansion cannot be performed for all tissues and organs. This explains the growing interest in stem cells that are produced during the development of the embryo, then of the fetus and finally in adult tissues and organs. Different stem cells with different potential can be isolated and characterized (totipotent or mesenchymal of different origins, especially those present in tissues…). It is undeniable that bone marrow, adipose tissue or Wharton’s Jelly stem cells are of potential interest for clinical applications because they are easily separated and prepared, and no ethical problems are involved in their use.

This multidisciplinary approach allows deciphering and better understanding the molecular mechanisms of stem cells differentiation as well as their immunological properties. This led to significant advances in the cells function and their potential use in clinics, especially epigenetics that seems to play an increasing role in the regulation of these processes. More recently the extracellular vesicles originated from stem cells appeared as a promising alternate for their use for cell-free therapy. This will contribute to the possibility to repair or reconstruct several types of tissues or organs (cartilage, bone, tendons, liver, vascular, dental or nerve tissues…) whose function has been altered by diseases or aging, or to use as new drugs for pathologies such as cancers or degenerative diseases.

A large number of potential methods exists for each tissue or type of therapy. For example, the amounts of tissue produced by in vitro cultures are generally higher when three-dimensional porous supports are used when compared with monolayer cultures. Moreover, mechanical stress influences the differentiation of cells. Such changes are now considered as critical for understanding pathological mechanisms (osteoarthritis, inflammation, atherosclerosis, etc.). Biotherapies have also major applications in the field of cancer therapies (i.e., leukemia, melanoma, prostate cancer, etc.).

However, and despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. In other respects, some lineages of adult stem cells are capable of greater plasticity than those of their origin tissue. Nowadays, mesenchymal stem cells represent an enormous potential value for regenerative medicine. During the last 10 years, these multipotent cells have generated considerable interest. They showed to escape allogeneic immune response and to be capable of immunomodulatory activity. These properties may be of a great interest for the future of regenerative medicine, but today it is probably better to consider mesenchymal stem cells as a mixed population of progenitors rather than as homogeneous stem cells.

The regeneration of tissues is and will remain a challenge for the future development of cell therapy and tissue engineering. Many problems remain to be solved and scientific and technical knowledge is lacking to lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardized production, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations.

The 9^th France-China Symposium on “Stem cells and Regenerative Medicine” was held in Strasbourg (France), 2–4 October 2019 at the Faculty of Dental Surgery. Strasbourg, located in the east part of France, is the capital of Alsace and one of the of Europe capitals by the presence of the Europe Parliament and other European institutions. It is well known by its University, Research Centers and Medical University Hospital, and welcomes several Nobel prizes.

The aim of this symposium was to provide researchers, clinicians and students a comprehensive up-to-date overview of stem cells and potential medical applications in cellular and tissue engineering for the treatment of various chronic diseases. The main objective was to bring scientists together from various disciplines and experiences and to discuss the recent advances in the use and applications of stem cells.

The Symposium was organized around three complementary themes introduced by eminent scientists renowned in their fields; cell, tissue engineering, and clinical applications. Important complementary aspects such as ethic, and cell marketing were also presented.

This book puts together the manuscripts from the presented lectures of the Symposium which illustrates the challenges and the recent progress performed on the characterization of stem cells.

The Guest Editors would like to thank all participants and authors for their valuable cooperation, contribution and efforts. The significant support of the Region Grand Est is gratefully acknowledged, together with CNRS, University of Lorraine and University of Strasbourg.

Daniel George

CNRS-UMR 7357, ICUBE Laboratory, University of Strasbourg, France

Jacques Magdalou

CNRS-UMR 7365, Biopôle, Faculté de Médecine, 54500 Vandœuvre-lès-Nancy, France

Jean-François Stoltz

CNRS-UMR 7365, Biopôle, Faculté de Médecine, 54500 Vandœuvre-lès-Nancy, France

The two national Academies of Medicine and Technology reviewed recently the development of stem-cells based medical applications and the related industrial and regulatory challenges that remain to be overcome in order to make these science-derived innovations accessible to the patients. Some of these issues are presented and discussed.

Stem cells used in therapy include mainly hematopoietic stem cells (HSC) to treat aplasias, leukemias and hematological genetic diseases, and mesenchymal stem cells (MSC) produced in small quantities by the bone marrow, but also other tissues, to treat cardiac and cutaneous diseases thanks to their secretory properties of growth factors. A major step was taken with the use of embryonic (ESC) or induced pluripotent stem cells (iPS). The former are used, after isolation and differentiation, either in rare therapeutic purposes or for the in vitro screening of drugs. iPS are produced from adult cells after reprogramming and differentiation and utilized for the treatment of various diseases in autologous or allogeneic form, the second condition allowing only mass production. New lines of research are now in progress including the creation of organoids that are templates of many organs of the body and allow the process of cell organization and its perturbations to be analyzed. Creation of post-meiosis gametes (23 chromosomes) from iPS or ESC is intended to treat serious genetic diseases. Creation of human chimera by interspecies blastocyst complementation has also been studied in view of organ transplantation. It is only allowed for the implantation of human cells in animal blastocysts and prohibited for the reverse. Implantation in uterus of the modified embryos is prohibited by the French law.

Stem cells are a kind of cells with the ability of self-renewal and multi-directional differentiation potential. A variety of stem cells or progenitor cells have been shown to be efficacy in the treatment of some refractory diseases. The mesenchymal stem cells (MSCs) are derived from mesoderm and have the characteristics of differentiation into three germ layers and immune regulation, which means that these cells are suitable for either autologous or allogeneic treatment and are ideal cells for regenerative medicine. MSCs can be isolated from various tissue types, including the bone marrow, fat, and perinatal tissues. From the perspective of ethics, medicine and cell engineering, adult bone marrow and adipose MSCs cannot be used as a mass production source of conventional treatment. Perinatal tissue, including umbilical cord, amniotic fluid and placenta, is a rich source of MSCs with strong immunosuppressive and proangiogeneic activities. These stem cells bring new hope for disease treatment. This paper reviews the research progress of stem cells as novel technology, novel therapies and industrial model in the field of regenerative medicine.

Type II collagen is the major collagen protein in cartilage, synthesized as precursor forms (procollagens). Several splice variants of the gene encoding type II procollagen have been identified such as IIA and IIB isoforms. Interestingly, a shift from IIA to IIB transcripts has been reported to occur during cartilage development and during chondrogenic differentiation of mesenchymal stem cells in vitro. Thus, type IIB procollagen represents a reliable marker of chondrocyte differentiation. We characterized previously the first antibody (referred as anti-pNIIB52) able to selectively detect the IIB form of human type II procollagen in Western-blot or immunohistochemistry analysis. More recently, we used anti-pNIIB52 in flow cytometry to quantify chondrogenic induction of bone marrow-mesenchymal stem cells cultivated in agarose hydrogel, after release of the cells from the gel. Here, we use imaging flow cytometry and anti-pNIIB52 to visualize directly intracellular accumulation of type IIB procollagen in cells undergoing chondrogenesis. Our data together show that flow cytometry analysis using anti-pNIIB52 represents an efficient and rapid diagnostic tool of good chondrogenic conversion, at the cellular level.

Objective:

A large number of studies have suggested that low birth weight fetuses were susceptible to fetal-originated diseases in adulthood. The purpose of this study was to investigate whether the histone 3 Lysine 9 (H3K9) acetylation level of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) was an early warning marker for the susceptibility of multiple chronic diseases in adulthood.

Methods:

The epigenetic and expressional abnormality of 11β-HSD2 in human WJ-MSCs induced by a variety of prenatal adverse xenobiotic factors were analyzed by real-time quantitative PCR (RT-qPCR), chromatin immunoprecipitation (ChIP) and western blotting (WB). The expression of insulin-like growth factor 1 (IGF1) in human WJ-MSCs after overexpression or knockdown of 11β-HSD2 gene was analyzed by RT-qPCR. Finally, immunofluorescence and ChIP-PCR were used to analyze the H3K9 acetylation level and expression of 11β-HSD2 in the human umbilical cord with intrauterine growth retardation (IUGR).

Results:

The mRNA and protein expression of 11β-HSD2 in WJ-MSCs were decreased after treatment with caffeine, nicotine, and ethanol. The histone acetylation level in 11β-HSD2 promoter region (H3K9) was significantly reduced simultaneously. In the proliferation model of WJ-MSCs, 300 nM of cortisol promoted the gene expression of the IGF1 pathway, while 1200 nM inhibited the gene expression of the IGF1 pathway. The expression of IGF1 treated with cortisol at 300 nM was decreased after overexpression of 11β-HSD2, while the expression of IGF1 was increased after treatment with 1200 nM cortisol. After knockdown of 11β-HSD2, the expression of IGF1 was decreased after treatment with 300 nM cortisol, and IGF1 gene expression decreased further after treatment with 1200 nM cortisol. Besides, the expression and H3K9 acetylation level of 11β-HSD2 in the IUGR-derived human umbilical cord was also reduced.

Conclusion:

The decreased expression and H3K9 acetylation level of 11β-HSD2 in WJ-MSCs induced by multiple xenobiotics exposures may mediate the decreased expression of IGF1. The H3K9 acetylation level of 11β-HSD2 in the human umbilical cord might be an early warning biomarker for evaluating susceptibility to multiple chronic diseases in adulthood.

Almost all cells in the human body are subjected to mechanical stresses. These forces can vary from a few Pascals (shear stress) to some mega Pascals (on hip cartilage). It is now well known that mechanical forces have a decisive effect on cellular physiology. In 1880, W. Roux introduced the concept of functional adaptation; which can be defined as a quantitative autoregulation controlled by stimuli like mechanical forces. These stresses influence functionality and cellular metabolism and can lead to appropriate tissue remodelling by triggering a cascade of reactions (mechanotransduction), being the signal for the adaptation of cells and tissues. However, although the main biological effects of mechanical forces are well documented, the relation between mechanical forces and physiological phenomena is largely unknown. In this paper, some effects of mechanical stresses on different cells (mesenchymal stem cells, bone cells, chondrocyte, endothelial cells, vascular or muscular cells, etc.) are summarized.

Repair and reconstruction of large bone defects remain a significant challenge. Cell construct, containing mesenchymal stem cells (MSCs) and scaffold, is a promising strategy for addressing and treating major orthopedic clinical conditions. However, the design of an ideal cell construct for engineering bone faces two critical challenges (i) matching the scaffold degradation rate to that of new bone formation and (ii) preventing the massive cell death post-implantation (caused by disruption of oxygen and nutrient supply). We will hereby primarily focus on the challenge of survival of MSCs post-implantation. Increasing evidence indicates that metabolic regulation plays a critical role in cell fate and functions. In cell metabolism, glucose is considered the major metabolic substrate to produce ATP via glycolysis when the availability of oxygen is limited. In this paper, we delineate the essential roles of glucose on MSC survival. We aim to provide a different perspective which highlights the importance of considering glucose in the development of tissue engineering strategies in order to improve the efficiency of MSC-based cell constructs in the repair of large bone defects.

Orthodontic fixed appliances are used to correct dental malocclusions by optimizing tooth movement and associated bone remodelling. Currently, orthodontic archwires made of shape memory alloys (SMAs) are widely used to initiate these treatments. We conduct experiments on SMA wires in pseudo in-vivo conditions, complementary to ISO standards, to assess the influence of temperature and humidity and to highlight their expected mechanical behaviour for clinical use. For this, an in-house built measurement device was developed to carry out experiments at controlled temperatures (21°C and 35°C) and in dry or wet conditions (artificial saliva). The dental arch was reproduced by 3D printing. The results show that the temperature has a major influence on the delivered forces whereas wet or dry conditions seem to have less impact. Also, we emphasize that at 35°C (in mouth conditions), in wet or dry conditions, SMAs superelasticity is only effective for displacements up to about 3 mm when an entire dental arch is considered.

Background:

3D printing has become a promising tool for cartilage engineering, combining 3D deposition of cells seeded in supporting biomaterials.

Objective:

Our goal was to evaluate the chondrogenic properties of three different bioinks, seeded with human bone marrow mesenchymal stem cells (bMSCs).

Methods:

The three different tested bioinks are seeded with 2 × 10⁶ cells/mL bMSCs. The bioink#1 is composed of gelatin, fibrinogen, and very low viscosity alginate. The bioink#2 has the same composition, excepted for the alginate that is a low viscosity one. The bioink#3 is manufactured by CELLINK^®. The cartilaginous substitutes were cultivated for 28 days in the presence of ITS vs TGF-ß1. The extracellular matrix synthesis is evaluated at D28 by histology (Hematoxylin-Eosin-Saffron & Alcian Blue) and immunostaining (type II collagen).

Results:

The bioink#1 better promoted type II collagen synthesis, although the three bioink were equipotent in terms of proteoglycan content. Despite its universal characteristics, the bioink#3 failed to encourage the hyaline-like matrix synthesis.

Conclusion:

The bioink#1 containing gelatin, fibrinogen, and very low viscosity seems to be the fittest of the three bio-inks to obtain a cartilaginous substitute presenting a remarkable matrix synthesis. This study confirms the importance of the choice of bioink for cartilage engineering.

Irreversible pulp inflammation is so painful that the clinical treatment is the removal of the entire pulp tissue. The current irreversibility of this inflammation is due to the lack of suitable biomaterials able to control it and to orchestrate pulp regeneration. Vitality of the tooth is so important for its functional life that adequate regenerative biomaterials must be developed. Whatever the degree of tooth maturity and its pathology, pulp and surrounding tissues constitute a treasure of dental stem cells. Advances of regenerative nanomedicine provide innovative strategies to use these strongly regenerative stem cells for endodontic regeneration. These cells can support endodontic regeneration by cell homing or by being seeded in biomaterials. Whatever the regenerative strategy, nanotechnologies optimise the attraction, colonisation, proliferation and differentiation of dental stem cells. The nano-reservoirs of active biomolecules orchestrate and enhance their cellular functions. The nanofibers constitute biomimetic scaffolds which promote their pulp connective tissue regeneration. Nanostructured composite scaffolds functionalized by controlled drug delivery systems of several active biomolecules would be the future nanobiomaterials for meeting the challenge of the complex endodontic regeneration.

Obstacles persist in the treatment and prevention of articular cartilage defects. Polycaprolactone (PCL) and poly(vinyl-pyrrolidone) (PVP) biomaterials were obtained by electrospinning and electrospraying to inspect their potential application for cartilage regeneration. Sodium hyaluronate (SH) was then added into nanofibers of PCL and particles of PVP. The aim of incorporating sodium hyaluronate to this polymer is to enhance the capacity of articular cartilage to regenerate. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were seeded onto these tissue engineering (TE) products. The cell viability in vitro and the ability of biomaterials to support the chondrogenic differentiation of hBM-MSCs have been assessed. We report here that hBM-MSCs on these biomaterials were not able to regenerate articular cartilage mainly due to unsuitable culture environment.

Research purposes:

To explore the clinical effect of local injection of autologous platelet-rich plasma (platelet-rich plasma) for lateral epicondylitis of humerus.

Research method:

A total of 58 patients with lateral epicondylitis of the humerus who were treated in the orthopedics department of our hospital from January 2018 to December 2018 were selected and included in the study. Subjects were treated with local injection of autologous platelet-rich plasma. Before the treatment, at 1 week, 1 month and 3 months after the treatment, the subjects were evaluated with visual analogue scale (VAS) and Mayo elbow function score (MEPS) for pain and elbow function.

Results:

Compared with before treatment, VAS decreased and MEPS increased after treatment. The four indicators of MEPS—pain, activity, stability, and daily abilities were significantly improved.

Conclusion:

Local injection of autologous PRP in the treatment of lateral epicondylitis of the humerus can better relieve pain symptoms and improve elbow joint function.

Urothelium is a highly specialized epithelium covering the entire urinary excretory system. Tissue engineering of this urinary tract may allow to consider its reconstruction to perform in vitro studies or in vivo replacement. Therefore, the question of specific reconstruction of the urothelium arises in order to guarantee the neotissue’s ability to act as a barrier against highly cytotoxic urine. This literature review describes the different cell types and strategies available for this reconstruction. The non-reconstruction of urothelium relies on the colonization of a biomaterial by the adjacent healthy tissue but allows only incomplete reconstruction and fibrosis. The use of autologous urothelial cells requires preliminary surgery and has not been successful enough in humans. Research has therefore focused on the use of stem cells. Adipose Derived Stem Cells (ADSCs) and Bone Marrow Derived Stem Cells (BMSCs) allow the reconstruction of the smooth muscle layer, but have little effect on urothelium reconstruction. Urine Derived Stem Cells (UDSCs) and Bladder mesenchymal Stem Cells (BSCs) are very promising because they allow the achievement of a differentiated urothelium. Induced Pluripotent Stem Cells (IPSCs) and Embryonic Stem Cells (ESCs) can be differentiated towards urothelial phenotype but their use is restricted by ethics.

Electrical stimulation (ES) can promote peripheral nerve repair. Nevertheless, the basis of ES generally requires conductive tissue engineering scaffolds. In this work, a neural tissue engineering scaffold is prepared from a series of conductive composites. The conductive composites, hydroxyethyl cellulose (HEC)/soy protein isolate (SPI)/polyaniline (PANI) films (HSPFs), were prepared by natural volatilization of HEC/SPI solution and then in-situ polymerization of aniline. Subsequently, the HSPFs films were confirmed by ATR-FTIR, water contact angle and SEM characterization. The conductivity of HSPFs reached 0.45 S/m superlatively and cell contact test showed that HSPFs had good cytocompatibility with PC12 cells. Most important of all, the neurite lengths and BDNF protein expression of PC12 cells on HSPFs can be promoted by ES. These results indicated that the ES may have potential application in nerve tissue engineering field through the conductive HSPFs films.

The invasive measurement of the hepatic venous pressure gradient is still considered as the reference method to assess the severity of portal hypertension. Even though previous studies have shown that the liver stiffness measured by elastography could predict portal hypertension in patients with chronic liver disease, the mechanisms behind remain today poorly understood. The main reason is that the liver stiffness is not specific to portal hypertension and is also influenced by concomitant pathologies, such as cirrhosis. Portal hypertension is also source of a vascular incidence, with a substantial diversion of portal venous blood to the systemic circulation, bypassing the liver. This study focuses on this vascular effect of portal hypertension. We propose to generate and control the portal venous flow (to isolate the modifications in the portal venous flow as single effect of portal hypertension) in an anesthetized pig and then to quantify its implications on liver stiffness by an original combination of MRE and 4D-Flow Magnetic Resonance Imaging (MRI). A catheter balloon is progressively inflated in the portal vein and the peak flow, peak velocity magnitude and liver stiffness are quantified in a 1.5T MRI scanner (AREA, Siemens Healthcare, Erlangen, Germany). A strong correlation is observed between the portal peak velocity magnitude, the portal peak flow or the liver stiffness and the portal vein intraluminal obstruction. Moreover, the comparison of mechanical and flow parameters highlights a correlation with the possibility of identifying linear relationships. These results give preliminary indications about how liver stiffness can be affected by portal venous flow and, by extension, by hypertension.

Tissue engineering is a method of constructing seeding cells and artificial materials as the cytoskeleton in vitro, in order to fabricating artificial organs and tissues. Reconstruction of corneal epithelial tissue in vitro by tissue engineering technique brought hope to the corneal blind patients. In this study, we used human umbilical cord mesenchymal stem cells (hUC-MSCs) as seeding cells, and cross-linked amniotic membrane by genipin as the cytoskeleton to reconstructing corneal epithelial tissue in vitro. In addition, we tested the tenacity, hardness, degradation speed, cytocompatibility and inflammatory response in preclinical application of this new artificial material, for the purpose of finding a new approach of modifying amniotic membrane close to the feature of natural cornea. As a result, the best cross-link condition-1.0% genipin cross-linked with amniotic membrane under 45°C for 24 hr could improve the physical character of natural amniotic membrane. Genipin cross-linking makes amniotic membrane and seeded hUC-MSCs has better cytocompatibility and lower inflammatory response in preclinical application.

Objectives:

Statins have been proposed as interesting pharmacological treatment for periodontal diseases because of their pleiotropic effect. Statins modulate bone metabolism, immuno-inflammatory complex and bacterial clearance. However, their systemic administration is associated to side effects. Therefore, their local administration has been suggested. The aim of this study was to evaluate the potential pro-regenerative effects of a thermosensitive gel functionalized by lovastatin on Porphyromonas gingivalis elicited inflammation in vitro and bone regeneration in vivo.

Methods:

Physical and chemical properties of a thermosensitive lovastatin loaded chitosan gel were evaluated. The anti-inflammatory effect of lovastatin was assessed in vitro by RT-qPCR and Elisa. In vivo, a model of calvarial defect was used to confirm the pro-regenerative effect on periodontal wound healing.

Results:

In vitro, lovastatin was able to decrease TNF-α secretion in P.gingivalis stimulated cells (unmapped: inline-formula unmapped: math unmapped: mi punmapped: mo <unmapped: mn 0.05). In vivo, local application of chitosan gel functionalized with lovastatin improved wound healing at calvarial site in comparison with untreated controls and mice treated with systemic statin administration.

Conclusions:

This study demonstrates the potential regenerative effects of local application of a thermosensitive gel functionalized by lovastatin.

Drawing on our clinical expertise with diabetic patients and on a retrospective study focused on patients with foot ulcers or wounds and Peripheral Artery Disease, we show a healing problem exists specifically in diabetic patients, despite arterial revascularization. To overcome this specific problem, Cell Therapy could be a way, exclusively aimed at diabetic patients. We explain the reasons why, as well as the ways and means, and more particularly the concept of tissue reversibility.